Method of processing nucleic acid sample

Abstract

The present disclosure provides methods and systems for amplifying and analyzing nucleic acid samples. The present disclosure provides methods for preparing cDNA and / or DNA molecules and cDNA and / or DNA libraries using modified reverse transcriptase enzymes.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 672,480, filed on May 16, 2018, and U.S. Provisional Application No. 62 / 779,371, filed on December 13, 2018, the contents of each of which are incorporated herein by reference in their entirety. Background Art

[0003] A common technique for studying gene expression in living cells is to produce complementary deoxyribonucleic acid (cDNA) from ribonucleic acid (RNA) molecules. This technique provides a way to study RNA from living cells, which avoids directly analyzing RNA, which is unstable in itself. As the first step in cDNA synthesis, RNA molecules from an organism are separated from an extract of an organism's cells or tissues. After separating the messenger RNA (mRNA), the oligonucleotide sequence is annealed to the separated mRNA molecule using methods such as affinity chromatography using oligo dT (a short sequence of deoxythymidine nucleotides), and the RNA / DNA primer can be used as a template to produce a cDNA copy of the RNA sequence using an enzyme with reverse transcriptase activity.

[0004] Therefore, reverse transcription of mRNA is a key step in many forms of gene expression analysis. Typically, mRNA is reverse transcribed into cDNA for subsequent analysis by primer extension or polymerase chain reaction.

[0005] Reverse transcriptase has both RNA-guided DNA polymerase activity and DNA-guided DNA polymerase activity. Reverse transcription of RNA templates may require primer sequences that are annealed to the RNA template to initiate DNA synthesis from the 3'OH of the primer. At room temperature, reverse transcriptase can allow the formation of fully matched and unmatched DNA / RNA hybrids. In some cases, reverse transcriptase may produce a large amount of nonspecific cDNA products due to such nonspecific initiation events. Nonspecific reverse transcriptase products can interfere with subsequent cDNA analysis, such as cDNA sequencing, real-time polymerase chain reaction (PCR) and alkaline agarose gel electrophoresis, etc. The nonspecific cDNA templates produced by nonspecific reverse transcriptase activity may present specific difficulties in applications such as real-time PCR. Specifically, such nonspecific cDNA products may produce erroneous signals, which complicates the analysis of real-time PCR signals and products. Therefore, reducing nonspecific reverse transcriptase activity can make cDNA synthesis more specific. At present, there is no reliable and easy-to-use method for improving reverse transcription specificity. The present disclosure meets these and other needs.

[0006] Several methods can be used to obtain transcriptome data from single cells. A pioneer method uses reverse transcriptase and oligo-dT primers, in which the T7 phage RNA polymerase promoter sequence is connected to the 5' end of the oligo-dT operation. The resulting cDNA is transcribed into multiple RNA copies and then converted back to cDNA (Phillips et al., Methods 10 (3): 283-288 (1996)). This often truncates the cDNA molecule, thereby losing the 5' sequence of the original mRNA, especially for relatively long transcripts, and when using lower amount (LQ) cells as starting materials, multiple rounds of processing are required, which further aggravates cDNA truncation. Recent modifications (Hashimshony et al., Cell Rep. 2 (3): 666-673 (2012)) enable multiplex analysis, but this still favors 3' terminal sequences. Other methods are based on PCR amplification of cDNA (Liu et al., Methods Enzymol. 303:45-55 (1999); Ozsolak et al., Genome Res. 20(4):519-525 (2010); Gonzalez et al.,), PLoS ONE (5(12):el4418 (2010); Kanamori et al., Genome Res. 21(7):1150-1159 (2011); Islam et al., Genome Res. 20(4):519-525 (2010); Gonzalez et al.,), PLoS ONE (5(12):el4418 (2010); Kanamori et al., Genome Res. 21(7):1150-1159 (2011); Islam et al., Genome Res. 20(4):519-525 (2010); Gonzalez et al.,), PLoS ONE (5(12):el4418 (2010); Kanamori et al., Genome Res. 21(7):1150-1159 (2011); =(43)-49; (in Chinese) : (i) 5-6; (ii) 2-8; (iii) 3-11; (iv) 5-12; (v) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8; (vi) 1-6; (vi) 1-8

[0007] However, these methods may result in biased representation of sequences along the mRNA and fail to obtain the complete sequence of the mRNA (eg, long mRNA) due to exclusion of DNA templates (eg, long DNA templates) even when long PCR reactions are used. Summary of the invention

[0008] In some aspects, the present disclosure provides a method for producing a non-naturally occurring enzyme, the method comprising: a) expressing a heterologous sequence encoding the non-naturally occurring enzyme in a host, wherein the non-naturally occurring enzyme comprises: a first domain derived from an R2 retrotransposon, such as a finger domain; a second domain derived from an R2 retrotransposon, such as a thumb domain; a third domain derived from an R2 retrotransposon, such as a palm domain; and an endonuclease domain derived from an R2 retrotransposon; b) purifying the non-naturally occurring enzyme from the host, thereby producing the non-naturally occurring enzyme. In some cases, the non-naturally occurring enzyme further comprises a fusion tag molecule. In other cases, the fusion tag molecule stabilizes the non-naturally occurring enzyme and the fusion tag molecule is selected from the group consisting of: Fh8, MBP, NusA, Trx, SUMO, GST, SET, GB1, ZZ, HaloTag, SNUT, Skp, T7PK, EspA, Mocr, Ecotin, CaBP, ArsC, IF2-domain I, IF2-domain I derived tag, RpoA, SlyD, Tsf, RpoS, PotD, Crr, msyB, yjgD, rpoD and His6. In other cases, the fusion tag molecule is selected from the group consisting of: His tag, His6 tag, calmodulin tag, CBP, CYD (covalent but dissociable NorpD peptide), Strep II, FLAG tag, HA tag, Myc tag, S tag, SBP tag, Softag-1, Softag-3, V5 tag, Xpress tag, Isopeptag, SpyTag, B, HPC (protein C heavy chain) peptide tag, GST, MBP, biotin, biotin carboxyl carrier protein, glutathione-S-transferase tag, green fluorescent protein tag, maltose binding protein tag, Nus tag, Strep tag and thioredoxin tag. In some cases, at least one of the first domain, the second domain, the third domain or the endonuclease domain is derived from an arthropod. In some cases, at least one of the first domain, the second domain, the third domain or the endonuclease is derived from a vertebrate, an echinoderm, a flatworm, a hydra or a silk moth. In some cases, the non-naturally occurring enzyme has at least 90% identity to SEQ ID NO: 1-20. In some aspects, the host is selected from bacteria, yeast, algae, cyanobacteria, fungi, plant cells, Escherichia coli, or any combination thereof. In some cases, the non-naturally occurring enzyme comprises a mutagenic motif-1 sequence. In some cases, the mutagenic motif-1 sequence has improved skipping activity compared to the wild-type sequence. In some cases, the non-naturally occurring enzyme comprises a mutagenic motif 0 sequence. In some cases, the mutagenic motif 0 sequence has improved skipping activity compared to the wild-type sequence. In some cases, the non-naturally occurring enzyme comprises a mutagenic thumb sequence.In some cases, the mutagenized second domain sequence has improved single-strand priming efficiency or improved processivity.

[0009] In some cases, the present disclosure provides a non-naturally occurring enzyme comprising: (i) a first domain derived from an R2 retrotransposon, such as a finger domain; (ii) a second domain derived from an R2 retrotransposon, such as a thumb domain; (iii) a third domain derived from an R2 retrotransposon, such as a palm domain; and (iv) an endonuclease domain derived from an R2 retrotransposon. In some cases, the non-naturally occurring enzyme has at least 80% identity to SEQ ID NOs: 1-20.

[0010] In some cases, the present disclosure provides a method for simultaneously amplifying ribonucleic acid (RNA) molecules and deoxyribonucleic acid (DNA) molecules, comprising: (a) providing a reaction mixture comprising the RNA, DNA, and a non-naturally occurring enzyme, each of the non-naturally occurring enzymes comprising: (i) a first domain derived from a non-retroviral transposon or from an R2 retrotransposon, such as a finger domain; (ii) a second domain derived from an R2 retrotransposon, such as a thumb domain; (iii) a third domain derived from an R2 retrotransposon, such as a palm domain; and (iv) an endonuclease domain derived from an R2 retrotransposon; and (b) subjecting the reaction mixture to conditions sufficient to amplify the RNA and the DNA, thereby obtaining amplification products of the RNA and the DNA. In some cases, the DNA is a complementary DNA derived from a subset of the RNA in the reaction mixture.

[0011] In some cases, the present disclosure provides a method for preparing complementary deoxyribonucleic acid (cDNA) molecules, comprising: (a) partitioning a cell and a non-naturally occurring reverse transcriptase, the cell comprising ribonucleic acid (RNA) molecules; (b) releasing the RNA molecules from the cells in the partition; and (c) in the partition, synthesizing a complementary deoxyribonucleic acid (cDNA) library from the RNA molecules using the non-naturally occurring reverse transcriptase, the non-naturally occurring transcriptase synthesizing the cDNA library with a processivity of 20 nucleotides or longer nucleotides. In some cases, the non-naturally occurring reverse transcriptase has at least 80% identity to SEQ ID NO: 1-20. In some cases, the partition further comprises: i) one or more receptor nucleic acid molecules; and ii) a non-naturally occurring reverse transcriptase, wherein the non-naturally occurring reverse transcriptase has at least 80% identity to SEQ ID NO: 1-20.

[0012] In some aspects, the partition is a reaction space or cavity, which can be a droplet, a hole or a tube. In some cases, the droplet can be formed by contacting a first phase with a second phase that is immiscible with the first phase, such as contacting an aqueous phase with an oil phase.

[0013] In some cases, the present disclosure provides a method for processing a sample containing different types of ribonucleic acids (RNA), comprising synthesizing complementary deoxyribonucleic acid (cDNA) molecules using ribosomal ribonucleic acid (rRNA) molecules in the presence of ribosomal ribonucleic acid (rRNA) molecules that are blocked for transcription, such that less than 30% of the cDNA molecules contain sequences from the rRNA molecules. In some cases, the rRNA may not have been degraded and may be present during reverse transcription.

[0014] In some cases, the present disclosure provides a method for processing a mixture comprising ribonucleic acid (RNA) molecules, comprising: (a) fragmenting the RNA molecules in the mixture to obtain a plurality of RNA fragments; (b) contacting the plurality of RNA fragments with one or more single-stranded nucleic acid sequences, the one or more single-stranded nucleic acid sequences being complementary to at least a subset of the RNA fragments, thereby providing one or more RNA fragment complexes comprising the one or more single-stranded nucleic acid sequences hybridized to at least the subset of the RNA fragments; and (c) synthesizing at least one complementary deoxyribonucleic acid (cDNA) molecule from the RNA using a reverse transcriptase in the presence of the one or more RNA fragment complexes.

[0015] In some cases, the present disclosure provides a method for sequencing a single-stranded nucleic acid molecule, comprising: (a) providing a reaction mixture comprising the single-stranded nucleic acid molecule and a non-naturally occurring enzyme, wherein the non-naturally occurring enzyme comprises: a first domain derived from an R2 retrotransposon, such as a finger domain; a second domain derived from an R2 retrotransposon, such as a thumb domain; a third domain derived from an R2 retrotransposon, such as a palm domain; and an endonuclease domain derived from an R2 retrotransposon; (b) subjecting the reaction mixture to conditions sufficient to incorporate individual nucleotides into a growing chain complementary to the nucleic acid molecule using the non-naturally occurring enzyme, wherein the incorporation of the individual nucleotides into the growing chain yields a detectable signal; and (c) detecting the detectable signals, thereby sequencing the single-stranded nucleic acid molecule. In some aspects, the single-stranded nucleic acid molecule is an RNA molecule or a single-stranded DNA molecule. In some aspects, the conditions sufficient to directly sequence the single-stranded nucleic acid molecule include single-molecule sequencing conditions based on optics. In some aspects, the conditions sufficient to directly sequence the single-stranded nucleic acid molecule include microscopy-based single-molecule sequencing conditions. In some aspects, the conditions sufficient to directly sequence the single-stranded nucleic acid molecule include nanopore-based single-molecule sequencing conditions. In some aspects, the conditions sufficient to directly sequence the single-stranded nucleic acid molecule include field effect transistor-based single-molecule sequencing conditions.

[0016] In some aspects, the present disclosure provides a method comprising: (a) preparing complementary deoxyribonucleic acid (cDNA) molecules from one or more ribonucleic acids (RNA) using non-naturally occurring enzymes, wherein the one or more ribonucleic acids (RNA) are derived from an in situ tissue of a subject or from a fixed ex vivo tissue of the subject, wherein the non-naturally occurring enzyme comprises: a first domain derived from an R2 retrotransposon, such as a finger domain; a second domain derived from an R2 retrotransposon, such as a thumb domain; a third domain derived from an R2 retrotransposon, such as a palm domain; and an endonuclease domain derived from an R2 retrotransposon; thereby generating cDNA molecules from the in situ tissue of the subject or from the fixed ex vivo tissue of the subject; and (b) sequencing the cDNA molecules generated in (a). In some aspects, the fixed ex vivo tissue of the subject is fixed in formaldehyde or paraffin.

[0017] In some aspects, the present disclosure provides a method for preparing complementary deoxyribonucleic acid molecules, comprising: (a) fragmenting ribonucleic acid molecules to obtain multiple RNA fragments; (b) removing 3'-phosphate groups, 2'-phosphate groups and cyclic 2'3' phosphates from one or more of the RNA fragments, thereby producing one or more dephosphorylated fragmented RNAs; (c) adding a poly-A tail to the one or more dephosphorylated fragmented RNAs; (d) adding the following to the one or more dephosphorylated fragmented RNAs: a primer adapter comprising an oligo-T sequence; an acceptor adapter; and a non-naturally occurring R2 enzyme having a processivity of 20 nucleotides or longer nucleotides, wherein the non-naturally occurring R2 enzyme reverse transcribes a sequence from the one or more dephosphorylated fragmented RNAs in a 3' to 5' order, wherein the R2 enzyme jumps to the 3'-end of the acceptor adapter when reaching the 5' end of the one or more dephosphorylated fragmented RNAs. In some aspects, the acceptor adapter comprises a nucleotide analog. In some aspects, the nucleotide analog is at the 5' end of the acceptor adapter, the primer adapter, or both. Some aspects further comprise removing one or more unannealed primer adapters of (d)(i) prior to adding the non-naturally occurring R2 enzyme. In some cases, the one or more unannealed primer adapters are removed with an immobilized poly A oligomer. In some aspects, the acceptor adapter comprises a 3'-dideoxynucleotide at the 3'-end of the acceptor adapter.

[0018] In some aspects, the present disclosure provides a method for preparing complementary deoxyribonucleic acid molecules, comprising: (a) fragmenting a ribonucleic acid (RNA) molecule to obtain a plurality of fragmented RNA fragments; (b) adding the following to the one or more fragmented RNAs: a primer adapter; an acceptor adapter; and a non-naturally occurring R2 enzyme having a processivity of 20 nucleotides or longer nucleotides, wherein the non-naturally occurring R2 enzyme uses a plurality of ssDNA primers that are not complementary to a template to initiate reverse transcription, wherein upon reaching the 5' end of the fragmented RNAs, the R2 enzyme jumps to the 3'-end of the acceptor adapter.

[0019] In some aspects, the present disclosure provides a method for preparing complementary deoxyribonucleic acid molecules, comprising: a) fragmenting a ribonucleic acid (RNA) molecule to obtain a plurality of fragmented RNA fragments; (b) adding the following to the one or more fragmented RNAs: i. a primer adapter; ii. an acceptor adapter; and an enzyme, wherein the enzyme uses a plurality of ssDNA primers that are not complementary to the template to initiate reverse transcription, wherein upon reaching the 5' end of the fragmented RNAs, the enzyme jumps to the 3'-end of the acceptor adapter. In some cases, the enzyme is a non-naturally occurring R2 enzyme.

[0020] In some aspects, the present disclosure provides a method for preparing complementary deoxyribonucleic acid molecules, comprising: a) adding the following to unfragmented ribonucleic acid (RNA) molecules: i. a primer adapter; ii. an acceptor adapter; and iii. an enzyme, wherein the enzyme uses a plurality of ssDNA primers that are not complementary to the template to initiate reverse transcription, wherein upon reaching the 5' end of the fragmented RNA, the enzyme jumps to the 3'-end of the acceptor adapter. In some cases, the enzyme is a non-naturally occurring R2 enzyme.

[0021] In some aspects, the present disclosure provides a method for depleting multiple ribonucleic acids (RNAs) from a sample, comprising: (a) synthesizing complementary deoxyribonucleic acid (cDNA) molecules from a ribonucleic acid template from the sample; (b) incorporating a first adapter molecule into the 3' end of the synthesized cDNA molecule and incorporating a second adapter molecule into the 5' end of the synthesized cDNA; (c) performing up to 10 cycles of polymerase chain reaction (PCR) with a modified oligonucleotide probe complementary to the rDNA sequence, wherein the modified oligonucleotide probe is configured to allow the probe to bind to a solid support, thereby producing a hybridization product bound to the solid support in the reaction mixture; (d) removing the synthesized cDNA from the reaction mixture while the hybridization product is bound to the solid support, thereby depleting the multiple ribonucleic acids (RNAs) from the sample.

[0022] In some aspects, the present disclosure provides a method for depleting multiple ribonucleic acids (RNAs) from a sample, comprising: (a) synthesizing an asymmetric double-stranded deoxyribonucleic acid molecule that is protected from enzymatic degradation at a first 5' end and is unprotected at a second 5' end from a ribonucleic acid (RNA) molecule under conditions sufficient to allow synthesis of the asymmetric double-stranded deoxyribonucleic acid molecule by adding the following to a reaction vessel containing the RNA molecule: i. a primer, wherein the primer comprises a modification at its 5' end that is configured to prevent enzymatic degradation by a 5' to 3' exonuclease; and ii. an enzyme; (b) adding the 5' to 3' exonuclease to the product of step (a), thereby producing ssDNA having a predetermined polarity; and (c) depleting the multiple RNAs from the product of (b) by hybridizing one of a plurality of probes to the multiple RNAs and performing a pull-down reaction.

[0023] In some aspects, the present disclosure provides a method for preparing a sample for RNA sequencing, comprising: (a) individually labeling a plurality of single cells with a plurality of unique barcodes; (b) combining the plurality of single cells in a single pot; (c) performing an RNA sequencing reaction on the plurality of single cells; (d) selecting a cell of interest based on the RNA sequencing reaction and identifying a unique barcode associated with the cell of interest, wherein the selection and the identification are performed in a computer program product; and (e) hybridizing a primer and the unique barcode associated with the cell of interest and performing an amplification reaction in the hybridized sample, thereby generating a plurality of amplicons.

[0024] In some aspects, the present disclosure provides a method for preparing a sample for ribonucleic acid (RNA) sequencing, comprising: (a) dissociating a tissue sample in a lysis reaction, thereby generating a plurality of nucleic acid templates from the dissociated sample, wherein the nucleic acid templates comprise ribonucleic acids; (b) synthesizing complementary deoxyribonucleic acid (cDNA) molecules from the plurality of nucleic acid templates from the dissociated sample, wherein the cDNA molecules are synthesized using a non-naturally occurring R2 enzyme having a processivity of 20 nucleotides or longer, wherein the synthesis is performed in the presence of one or more reagents used in the lysis reaction or in the presence of a plurality of cell fragments from the tissue sample.

[0025] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. It should be appreciated that the present disclosure is capable of other and different embodiments, and that its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Therefore, the drawings and description should be regarded as illustrative in nature and not restrictive.

[0026] Incorporated by Reference

[0027] All publications, patents and patent applications, and NCBI accession numbers mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, patent application, or NCBI accession number was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents, patent applications, or NCBI accession numbers incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The novel features of the present disclosure are set forth in detail in the appended claims. The features and advantages of the present disclosure will be better understood by reference to the following detailed description and accompanying drawings (hereinafter also referred to as "Figures" and "FIG.") which illustrate illustrative embodiments utilizing the principles of the present disclosure, in which:

[0029] Figure 1A The R2 N-terminal domain is shown. This figure shows a comparison of conserved sequence motifs in the amino-terminal domain of the R2 element. This figure also highlights the CCHH zinc finger and KWRK c-myb DNA binding motifs. The continuation of the N-terminal domain alignment is shown in Figure 1B.

[0030] Figure IB shows the R2 N-terminal domain. This figure is from Figure 1A Continuation of the alignment, therefore, also shows the comparison of conserved sequences in the N-terminal domain of the R2 element. The following sequence at the C-terminus is the RT and endonuclease domain, which is not shown in this figure.

[0031] Figure 1C Schematic diagram showing the amino acid sequence similarity of R2 elements of nine arthropods, with the N-terminal domain labeled.

[0032] Figure 2 R2 reverse transcriptase is shown. This figure shows a comparison of the conserved sequence motifs of the N-terminal portion of the reverse transcriptase R2 element. This figure highlights motif-1 and motif 0. Both motifs are subjected to engineering / mutagenesis in the methods of the present disclosure.

[0033] Figure 3 The R2 reverse transcriptase (RT) thumb domain is shown. This figure shows a comparison of conserved sequence motifs. The R2 RT thumb domain was subjected to engineering / mutagenesis in the methods of the present disclosure.

[0034] Figure 4 The R2 reverse transcriptase (RT) thumb domain is shown. Figure 3 Continuation of and showing comparison of conserved sequence motifs. In the methods disclosed herein, the R2 RT thumb domain was subjected to engineering / mutagenesis.

[0035] Figure 5A The method of rRNA sequence depletion as described in the present disclosure is shown, while rRNA depletion is integrated into the sample preparation process. This figure shows the different steps of the procedure, wherein ssDNA complementary to rRNA (DNA-sponge) is included in the library preparation reaction during or immediately after RNA sample fragmentation. DNA-sponge is a large ssDNA fragment that is at least partially complementary to the sequence of an rRNA subunit.

[0036] Figure 5B As described in this disclosure and Figure 5A The rRNA sequence depletion method shown in . However, Figure 5B A method is shown in which an RCA product complementary to rRNA is used instead of circular ssDNA (DNA-sponge).

[0037] Figure 6Shows the ability of in situ RNA sequencing to allow whole genome profiling of gene expression in situ in fixed cells and tissues. This figure shows the steps of in situ RNA sequencing, where RNA is converted to cDNA and sequenced directly or converted into a sequencing library using a single molecule approach. This figure also shows a spatially specific barcoding technique, where a glass plate printed with barcoded primer oligonucleotides is used.

[0038] Figure 7 A general poly A-based approach to RNA sequencing library preparation is shown. The figure shows the different steps starting with RNA fragmentation. Methods for fragmentation include natural RNA magnesium-induced degradation or enzymatic cleavage. The figure shows that, depending on which method is used, cleavage can produce either a 3'-OH or a cyclic 2',3'-phosphate at the 3'-end of the RNA fragment.

[0039] Figure 8 Show Figure 7 This figure shows a method in which an engineered T4 PNK is used, thus allowing the simultaneous use of the enzymes T4 PNK and poly A polymerase, rather than Figure 7 The program is divided into two steps.

[0040] Fig. 9 The library products and adapter dimer artifacts that can be generated in the reaction with the R2 enzyme are shown. The adapter dimer artifacts including the extension of the acceptor are prevented by 3'-dideoxynucleotides at the 3'-end of the acceptor adapter (alternatively, different extension blockers such as 3' phosphorylated-dNTP, 3' amino-dNTP can be applied). Artifacts caused by primer adapters (including poly-T sequences) are removed from the reaction, where oligo-A is connected to magnetic beads. Artifacts are caused without annealing (forming a template primer duplex), so the primer sequence remains single-stranded.

[0041] Fig.10 3'-end priming and extension using ssDNA primer adapter and R2 is shown. In this mechanism, extension is initiated by the ssDNA primer on the 3'-end of the template, and the complementary sequence does not anneal to the template. This figure shows that the library product is a full-length copy of the template.

[0042] Fig.11The method of random priming by random fragmentation is shown. This figure shows the first step, in which the RNA sample is fragmented. This figure also shows the second step, in which the RNA sample is mixed with a primer adapter (ssDNA), an R2 enzyme, and an acceptor adapter (ssDNA or RNA). The figure shows the third step, which is cleanup by solid phase reversible immobilization (SPRI), in which size selection is used to remove some adapter-adapter dimer artifacts. The figure shows the previous step, polymerase chain reaction (PCR) amplification, using primers that are complementary to both the primer adapter and the acceptor adapter.

[0043] Fig.12 Show Fig.11 Same method as described in , however, with a different cleanup reaction. This figure shows the fragmentation steps, 3'-end priming and a final cleanup reaction where oligomeric magnetic beads complementary to the ssDNA adapters are used to remove adapter dimers and artifacts.

[0044] Fig.13 Shown is a phylogenetic tree highlighting the inferred evolutionary relationship between the non-LTR retroelements and Bombyx mori, here with a minimum of 27% identity, and the R2 retrotransposon centered on the fragment of the RT-endonuclease.

[0045] Fig.14 The Bombyx mori R2 reference sequence (SEQ ID NO: 97) is shown.

[0046] Fig.15 Motif-1 point mutations are shown.

[0047] Fig.16 Motif 0 point mutation is shown.

[0048] Fig.17 Thumb subunit substitutions are shown, Fig.18 Continue.

[0049] Fig.18 The thumb subunit substitution is shown. Fig.17 The continuation of.

[0050] Fig.19 A method for removing certain artifacts is shown.

[0051] Fig. 20 A computer system is shown that is programmed or otherwise configured to implement the methods provided herein.

[0052] Fig.21 The steps of a bulk (rRNA) sequence depletion method using PCR extension and biotinylated oligoprimers are shown.

[0053] Fig. 22 The steps of a bulk (rRNA) sequence depletion method using 5'-end protected oligonucleotides (PCR primers) are shown.

[0054] Fig.23 Shown are steps of a method for sequencing a pool of single cells, where each single cell is labeled with a unique barcode as a selective target for PCR amplification.

[0055] Fig.24 Shown are the steps of a method for preparing RNA-seq libraries directly from tissue / cell biomass without RNA purification.

[0056] Fig.25 The bioanalyzer trace in Example 25 is shown.

[0057] Fig.26 The molecular structure of the library in Example 25 is shown. DETAILED DESCRIPTION

[0058] Although various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Those skilled in the art may conceive of numerous variations, changes, and substitutions without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present application including the definitions shall prevail. In addition, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Unless it is indicated that only a specific portion of a patent or patent publication is incorporated by reference, all publications, patents and other references mentioned herein are incorporated by reference for all purposes, just as each individual publication or patent application is specifically and individually indicated to be incorporated by reference. In order to further define the present disclosure, the following terms, abbreviations and definitions are provided.

[0060] The term "about" generally refers to variations in quantity that may occur, for example, due to typical measurement and liquid handling procedures used to prepare concentrates or solutions in the real world, inadvertent errors in these procedures, differences in the manufacture, source, or purity of the ingredients used to prepare the composition or perform the implementation method, and so on. The term "about" also encompasses amounts that differ due to different equilibrium conditions of the composition resulting from a specific initial mixture. Whether or not modified by the term "about", the claims include equivalents of such equivalents. In some cases, the term "about" means within 10% of the reported value, or within 5% of the reported value, or within 20% of the reported value.

[0061] The indefinite article "a" and "an" before the elements or components of the present disclosure are not intended to limit the number of instances, i.e., the number of occurrences of the elements or components. Therefore, "a" and "an" should be understood to include one or at least one, and unless the number obviously indicates the singular, the singular form of the elements or components also includes the plural.

[0062] As used herein, "non-LTR retrotransposon" generally refers to naturally occurring proteins encoded by non-LTR retrotransposon and polypeptide fragments thereof having reverse transcriptase activity, as well as proteins or polypeptides derived therefrom containing one or more amino acid substitutions that enhance reverse transcriptase activity or have no harmful effects. One type of non-LTR retrotransposon is the R2 protein or polypeptide. Therefore, as used herein, "R2 protein or R2 enzyme or polypeptide or a functional fragment thereof" refers to naturally occurring proteins encoded by R2 elements and polypeptide fragments thereof having reverse transcriptase activity, as well as proteins or polypeptides derived therefrom containing one or more amino acid substitutions that enhance reverse transcriptase activity or have no harmful effects.

[0063] As used herein, the terms "variant," "modified," "non-naturally occurring," and "mutated" are synonymous and refer to polypeptides or enzymes that differ from the specifically recited polypeptides or enzymes due to one or more amino acid insertions, deletions, mutations, and substitutions produced using, for example, recombinant DNA techniques (e.g., mutagenesis). Determining which amino acid residues can be substituted, added, or deleted without eliminating the associated activity can be guided by comparing the sequence of a particular polypeptide to homologous polypeptides (e.g., yeast or bacteria) and minimizing the number of amino acid sequence changes produced in regions of high homology (conserved regions) or by replacing amino acids with common sequences. In some cases, the terms "derived," "variant," "modified," "non-naturally occurring," and "mutated" are used interchangeably.

[0064] The terms "annealing", "hybridization" or "binding" generally refer to the combination of one or more single-stranded polynucleotide sequences, segments or chains, and allowing them to form a double-stranded molecule through base pairing. Two complementary sequences (e.g., ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA)) can anneal or hybridize by forming hydrogen bonds using complementary bases, thereby producing a double-stranded polynucleotide or a double-stranded region of a polynucleotide.

[0065] As used herein, the term "incorporating" when used in reference to adaptor incorporation may refer to the physical ligation of an adaptor, the extension of the adaptor, or the generation of a sequence complementary to the adaptor sequence in a nucleic acid molecule.

[0066] The term "subject" can be any animal that may benefit from the methods of the present disclosure, including, for example, humans and non-human mammals, such as primates, rodents, horses, dogs, and cats. Subjects include, but are not limited to, eukaryotes; mammals, such as primates, such as chimpanzees or humans, cows, dogs, cats; rodents, such as guinea pigs, rats, mice; rabbits; or birds; reptiles; or fish. Subjects particularly intended to be treated using the methods described herein include humans. A subject can be an individual or a patient.

[0067] As used herein, the term "primer extension reaction" generally refers to the denaturation of a double-stranded nucleic acid, the binding of a primer to one or both strands of the denatured nucleic acid, and the elongation of the primer.

[0068] As used herein, the term "reaction mixture" generally refers to a composition comprising reagents necessary to complete nucleic acid amplification (e.g., DNA amplification, RNA amplification), wherein non-limiting examples of these reagents include a primer set specific for a target RNA or target DNA, DNA produced by reverse transcription of RNA, DNA polymerase, reverse transcriptase (e.g., for reverse transcription of RNA), suitable buffers (including zwitterionic buffers), cofactors (e.g., divalent and monovalent cations), dNTPs, and other enzymes (e.g., uracil-DNA glycosylase (UNG)), etc. In some cases, the reaction mixture may also include one or more reporter agents.

[0069] As used herein, "reporter agent" generally refers to a composition that produces a detectable signal, the presence or absence of which can be used to detect the presence of an amplified product.

[0070] As used herein, the term "target nucleic acid" generally refers to a nucleic acid molecule in a starting nucleic acid molecule population having a nucleotide sequence for which presence, amount, and / or sequence, or one or more changes in presence, amount, and / or sequence need to be determined. The target nucleic acid can be any type of nucleic acid, including DNA, RNA, and analogs thereof.

[0071] The terms "polynucleotide", "nucleic acid", "nucleotide" and "oligonucleotide" are used interchangeably. These terms refer to polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides) of any length, fragments thereof or analogs thereof. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis (locus), exons, introns, messenger RNA (mRNA), transfer RNA, transfer-messenger RNA, ribosomal RNA, antisense RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), small interfering RNA (siRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If there is a modification to the nucleotide structure, it can be performed before or after assembling the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example, by binding to a labeling component. Nucleic acids described herein can contain phosphodiester bonds. In some cases, nucleic acids can be DNA (including, for example, genomic DNA, mitochondrial DNA, and cDNA), RNA (including, for example, mRNA and rRNA), or hybrids, wherein nucleic acids contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine (xathanine), hypoxathanine (hypoxathanine), isocytosine, isoguanine, etc. Polynucleotides are intended to cover single nucleic acids and multiple nucleic acids. Polynucleotides can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single-stranded and double-stranded DNA, DNA for a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA for a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA (can be single-stranded, or more typically double-stranded, or a mixture of single-stranded and double-stranded regions).

[0072] As used herein, the term "primer" refers to an oligonucleotide naturally occurring or synthetically produced in a restriction digest, characterized in that it can be extended relative to a template oligonucleotide, so that when all placed in a suitable temperature and pH in the presence of nucleotides, an oligonucleotide complementary to the sequence of at least a portion of the template molecule is connected to the primer. However, the ability to be used in this way does not require the primer to be fully extended relative to the template, and in some cases, the primer is only used as a site for adding a few non-templated nucleotides. For example, a primer of a primer hexamer of at least 6 nucleotides in length can be used. In some cases, a primer can be fluorescently labeled (e.g., 5'- / 56FAM / TGATGACGAGGCATTTGGC / 3'). In some cases, a primer has about 6 to about 100 nucleotides, or in some cases, a length in the range of about 10 to about 70 nucleotides. In some cases, a larger primer can be used. In some cases, a random primer can be used. In some cases, a primer can be a random primer. In some cases, one or more primers can be one or more random primers.

[0073] The term "one or more primers" can include any number of primers or random primers. For example, "one or more primers" can include at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 primers or random primers. The one or more primers can include about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 30, about 1 to about 35, about 5 to about 15, about 3 to about 10, about 5 to about 20, about 10 to about 50, about 30 to about 100, or more than about 100 primers. The one or more primers can include any number of primers.

[0074] As used herein, the term "random primer" refers to a primer having a random base sequence therein, and is intended to encompass primers consisting partially or entirely of a random base sequence.

[0075] As used herein, "homolog" refers to a protein that is functionally equivalent to an enzyme having an amino acid sequence with a designated sequence identification number (i.e., has the same enzymatic activity), but may have a limited number of amino acid substitutions, deletions, insertions or additions in the amino acid sequence. In order to maintain the function of the protein, the substitutions may be conservative substitutions, replacing one amino acid with an amino acid having similar properties.

[0076] The term "heterologous" refers to a molecule or activity derived from a source other than the reference species, while "homologous" refers to a molecule or activity derived from a host microorganism. Accordingly, exogenous expression of encoding nucleic acids of the present disclosure can use either or both heterologous or homologous encoding nucleic acids.

[0077] As used herein, the term "receptor template" generally refers to a nucleic acid molecule used to synthesize a complementary DNA (cDNA) molecule. The receptor nucleic acid may be modified. The receptor nucleic acid molecule may be modified at the 3' end, for example to protect it from being mistaken for an RNA primer. The modification of the receptor nucleic acid molecule may include a dideoxy 3' end. The modification may include a phosphorylated 3' end. In some cases, a polynucleotide or a phosphorylated 3' end of a receptor nucleic acid molecule that typically has a hydroxyl group at the 3' end may be used as a 3' blocker, because, for example, the extension performed by an enzyme or DNA polymerase disclosed herein may be inhibited or the joining performed by a ligase may be inhibited. Another non-limiting example of a 3' blocker includes adding a 3'C3 spacer (three-carbon spacer) to the 3' end of a polynucleotide, which may serve as an effective blocker for polymerase extension. Zhou et al., Clin. Chem., 50: 1328-1335 (2004). Thus, the 3' terminus can be blocked by the addition of, for example, a C3 spacer, a phosphate, an amino group (NH2), or any other chemical modification that inhibits the formation of a subsequent phosphodiester bond between the 3' terminus of the polynucleotide and another nucleotide.

[0078] As used herein, "overhang sequence" generally refers to a single-stranded region of a nucleic acid extending from a double-stranded region.

[0079] As used herein, an "isolated" polynucleotide generally refers to a polynucleotide that has been removed from its natural environment, produced using recombinant technology, or chemically or enzymatically synthesized. A polynucleotide may also be purified, i.e., substantially free of any other polynucleotides and associated cellular products or other impurities.

[0080] As used herein, the term "polymerase" generally refers to an enzyme that connects individual nucleotides together into a chain, using another chain as a template. In some cases, the polymerase is a polymerase with editing capabilities. In some cases, the polymerase with editing capabilities can be 3' to 5' exonuclease, T4 DNA polymerase, exonuclease I, Phi29, Pfu, Vent, KOD, exonuclease III and exonuclease T. Examples of polymerases can include DNA polymerases, RNA polymerases, RNA-guided DNA polymerases, reverse transcriptases, polypeptides having reverse transcriptase activity or any variants thereof, thermostable polymerases, wild-type polymerases, modified polymerases, Escherichia coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase, PHI 29 (phi29) DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tea polymerase, Tih polymerase, Tfi polymerase, Platinum polymerase, Taq polymerase, Tbr polymerase, Tfl polymerase, Tth polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, polymerase and variant with 3' to 5' exonuclease activity, modified product and derivative thereof. In some cases, polymerase can be reverse transcriptase or modified reverse transcriptase disclosed herein. In some cases, polymerase is a single subunit polymerase. The polymerase can have high continuous synthesis ability, i.e., the ability of polymerase to continuously incorporate nucleotides into nucleic acid template without stripping nucleic acid template. In some cases, polymerase can be the polymerase described in PCT / US2017 / 061197, such as P2.PCT / US2017 / 061197 (incorporated herein in its entirety).

[0081] As used herein, the term "reverse transcriptase" or RT generally refers to an enzyme having RNA-guided DNA polymerase and DNA-guided DNA polymerase. RT refers to a group of enzymes having reverse transcriptase activity (e.g., catalyzing the synthesis of DNA from an RNA template). In general, such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, reverse transcriptases plasmid reverse transcriptases, reverse transcriptases, bacterial reverse transcriptases, type II intron-derived reverse transcriptases, and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-long terminal repeat (LTR) retrotransposon reverse transcriptases, reverse transcriptases plasmid reverse transcriptases, reverse transcriptases, and type II intron reverse transcriptases. Other bacterial reverse transcriptases are described in Simon D and Zimmerly S (2008), "A diversity of uncharacterized retroelements in bacteria", Nucleic Acids Res, 36(22):7219-7229, and Kojima, KK and Kanehisa, M (2008), "Systematic survey for novel types of prokaryotic retroelements based on gene neighborhood and protein architecture", Mol Biol Evol, 25:1395-1404, which describe many classes of non-retroviral reverse transcriptases (i.e., retrocons, group II introns, and diversity-generating retroelements, etc.). Reverse transcriptase has been primarily used to transcribe RNA into cDNA, which can then be cloned into a vector for further processing or used in various amplification methods, such as polymerase chain reaction, nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), various primer extension reactions, 5'RACE, chemical modification detection, or other techniques that require the synthesis of DNA using an RNA template.

[0082] Reverse transcriptase

[0083] Reverse transcriptase can be separated from a large number of removable genetic elements from retrovirus and non-retrovirus sources. These removable genetic elements are present in the genome of higher species and play a role in the life cycle of these removable genetic elements. Removable genetic elements can encode the gene of reverse transcriptase (in Howard M Temin, "Reverse Transcription in the Eukaryotic Genome: Retroviruses, Pararetroviruses, Retrotransposons and Retrotranscripts (Reverse Transcription in the Eukaryotic Genome: Retroviruses. Pararetroviruses, Retrotransposons, and Retrotranscripts) ", "Molecular Biology and Evolution (Mol.Biol.Evol.) "2 (6): 455-468 in reviewed). These elements include but are not limited to retrotransposons. Retrotransposons include non-long terminal repeats (LTR) retrotransposons and LTR removable elements (such as TY3, TY5, non-LTR, LINE-L1, R2, R1). (Reviewed by Cordaux and Batzer in Nature Reviews, Vol. 10, pp. 691-703, October 2009).

[0084] Retroelements (genetic elements encoding RT) are divided into two major families represented as LTR-containing retroelements and non-LTR-containing retroelements (Xiong Y, Eickbush TH (1990) Origin and evolution of retroelements based upon their reverse transcriptase sequences (EMBO J) 9: 3353-62). Non-LTR retroelements are different families of RT coding elements, including retroplasmids, non-LTR retrotransposons, retrotransposons, and mobile type II introns.

[0085] The mutants disclosed herein can be produced according to any suitable method, including but not limited to the methods described and exemplified herein. For example, mutations of substitution, insertion, deletion and / or side chain modification can be introduced into the nucleotide and amino acid sequences of the relevant genes using any suitable technique (including site-directed mutagenesis) (Meth. Enzymol. 217, compiled by Wu, Academic Press (1993)). The λ Red recombinase method can be used to "knock out" genes (Datsenko et al., PNAS USA 97: 6640-6645 (2000)). Multiple gene disruptions without markers that last can be produced. Non-naturally occurring nucleotides and amino acids can also be used.

[0086] Methods for expressing non-naturally occurring enzymes from host systems

[0087] Non-natural enzymes, including R2 enzymes, may be difficult to manufacture, in part because of their size, structural complexity and amino acid composition. In addition, the R2 retroelement is a multi-domain element with a molecular mass usually exceeding 100 kD, which increases the challenge of manufacturing the R2 enzyme in a recombinant manner. In addition, the naturally occurring R2 enzyme in the host organism needs to be expressed at a low level, mostly because it has toxic effects on the host. In addition, it is believed that the naturally occurring R2 retroelement functions in a dimer form.

[0088] In some aspects, the present disclosure provides recombinantly produced enzymes comprising selected sequences derived from R2 retrotransposons; and vectors comprising nucleic acid sequences encoding recombinantly produced enzymes disclosed herein. The R2 retrotransposon element is generally composed of three main domains ( Figure 1A , 1B , 1C) consists of: N-terminal domain, reverse transcriptase domain and endonuclease domain. The N-terminal domain may include zinc fingers and c-myb DNA binding motifs, which are believed to affect the specific recognition and binding to the target DNA (target-triggered reverse transcription mechanism (TPRT). The reverse transcriptase domain is responsible for copying the R2 RNA template. Finally, the endonuclease domain is responsible for the specific cleavage of the target DNA.

[0089] In some aspects, the present disclosure provides a non-natural enzyme that is phylogenetically related to one or more elements of the R2 retrotransposon. In some cases, the enzyme of the present disclosure does not include the N-terminal domain of the R2 enzyme. In some aspects, the present disclosure provides a method for producing a non-natural enzyme, comprising: expressing a heterologous sequence encoding the non-natural enzyme in a host, wherein the non-natural enzyme comprises a first domain, a second domain, and a third domain derived from an R2 retrotransposon; For example, the palm, finger, and thumb domains derived from an R2 retrotransposon; The endonuclease domain derived from an R2 retrotransposon; And purifying the non-natural enzyme from the host, thereby producing the non-natural enzyme.

[0090] In some respects, the disclosure provides non-naturally occurring enzymes, wherein the N-terminal domain is removed. The N-terminal domain is considered to interfere with expression and stability of the R2 reverse transcription element. Thus, it is believed that the part of the entire N-terminal or the part of the N-terminal can improve expression and stability of the R2 reverse transcription element, without affecting the ability and performance of the disclosed enzyme in the RNA library prepared for sequencing. In some respects, the disclosure uses the sequence analysis of the R2 reverse transcription element related to R2 retrotransposon and other phylogenetics to identify the N-terminal domain. In some cases, the enzyme of the present disclosure is an enzyme related to one or more elements of the R2 reverse transcription element phylogenetically and in some cases, removes the N-terminal domain from the enzyme.

[0091] In some aspects, the present disclosure provides a non-naturally occurring enzyme comprising a fusion tag molecule, wherein the fusion tag molecule stabilizes the non-naturally occurring enzyme disclosed herein. In some aspects, the fusion tag molecule is selected from the group consisting of: Fh8, MBP, NusA, Trx, SUMO, GST, SET, GB1, ZZ, HaloTag, SNUT, Skp, T7PK, EspA, Mocr, Ecotin, CaBP, ArsC, IF2-domain I, IF2-domain I derived tags, RpoA, SlyD, Tsf, RpoS, PotD, Crr, msyB, yjgD, rpoD and His6.

[0092] In some aspects, the disclosure provides a non-natural enzyme comprising a fusion tag molecule, wherein the fusion tag molecule stabilizes the non-natural enzyme disclosed herein. In some aspects, the fusion tag molecule is selected from the group consisting of: His tag, His6 tag, calmodulin tag, CBP, CYD (covalent but dissociable NorpD peptide), StrepII, FLAG tag, HA tag, Myc tag, S tag, SBP tag, Softag-1, Softag-3, V5 tag, Xpress tag, Isopeptag, SpyTag, B, HPC (protein C heavy chain) peptide tag, GST, MBP, biotin, biotin carboxyl carrier protein, glutathione-S-transferase tag, green fluorescent protein tag, maltose binding protein tag, Nus tag, Strep tag and thioredoxin tag.

[0093] In some aspects, the disclosure provides a non-naturally occurring enzyme, wherein at least one of the palm and fingers domains from an R2 retrotransposon, the thumb domain of an R2 retrotransposon, or the endonuclease domain of an R2 retrotransposon is derived from an arthropod.

[0094] In some aspects, the disclosure provides a non-naturally occurring enzyme, wherein at least one of the palm and fingers domains from an R2 retrotransposon, the thumb domain of an R2 retrotransposon, or the endonuclease domain of an R2 retrotransposon is derived from Bombyx mori.

[0095] In some aspects, the disclosure provides a non-naturally occurring enzyme, wherein at least one of the palm and fingers domains from an R2 retrotransposon, the thumb domain of an R2 retrotransposon, or the endonuclease domain of an R2 retrotransposon is derived from a vertebrate or an echinoderm.

[0096] In some aspects, the disclosure provides a non-naturally occurring enzyme, wherein at least one of the palm and fingers domains from an R2 retrotransposon, the thumb domain of an R2 retrotransposon, or the endonuclease domain of an R2 retrotransposon is derived from a flatworm or a hydra.

[0097] In some aspects, the present disclosure provides a non-naturally occurring enzyme having at least 80% identity to SEQ ID No: 1-20, at least 81% identity to SEQ ID No: 1-20, at least 82% identity to SEQ ID No: 1-20, at least 83% identity to SEQ ID No: 1-20, at least 84% identity to SEQ ID No: 1-20, at least 85% identity to SEQ ID No: 1-20, at least 86% identity to SEQ ID No: 1-20, at least 87% identity to SEQ ID No: 1-20, at least 88% identity to SEQ ID No: 1-20, at least 89% identity to SEQ ID No: 1-20, at least 90% identity to SEQ ID No: 1-20, at least 91% identity to SEQ ID No: 1-20, at least 92% identity to SEQ ID No: 1-20, at least 93% identity to SEQ ID No: 1-20, at least 94% identity to SEQ ID No: 1-20, at least 96% identity to SEQ ID No: 1-20, at least 97% identity to SEQ ID No: 1-20, at least 98% identity to SEQ ID No: 1-20, at least 99% identity to SEQ ID No: 1-20, at least 100% identity to SEQ ID No: 1-10, at least 101% identity to SEQ ID No: 1-10 No: 1-20 is at least 94% identical, at least 95% identical to SEQ ID No: 1-20, at least 96% identical to SEQ ID No: 1-20, at least 97% identical to SEQ ID No: 1-20, at least 98% identical to SEQ ID No: 1-20, at least 99% identical to SEQ ID No: 1-20, at least 100% identical to SEQ ID No: 1-20.

[0098] In some aspects, the present disclosure provides non-naturally occurring enzymes, wherein the host is selected from bacteria, yeast, algae, cyanobacteria, fungi, plant cells, or any combination thereof. In some cases, the present disclosure provides non-naturally occurring enzymes, wherein the host is Escherichia coli.

[0099] In some aspects, the present disclosure provides a non-naturally occurring enzyme that can comprise one or more amino acid mutations in motif-1, motif-0, and thumb subunit ( Figure 2 , 3 and 4). Amino acid modifications suitable for improving the properties of R2-related enzymes can be conservative or non-conservative mutations. In some cases, motif-1 and motif-0 can be present in non-long terminal repeat (LTR) retrotransposons and telomerases, but not in retroviral transposons and LTR retroelements. Mutations can be made so that the encoded amino acids are modified to polar, non-polar, basic or acidic amino acids. Fig.15 and 16 Disclosed are possible mutations of motif-1 or motif-0 that can improve one or more properties of the R2 enzyme from silkworm moth, wherein Fig.14The Bombyx mori R2 reference sequence is disclosed in . For example, the following amino acid substitutions can be engineered in motif-1: A15→V15, A15→M15, A17→V17, A17→M17, H18→V18, H18→N18, R22→K22, R22→H22, Q23→123, Q23→E23, K24→A24, K24→124, K24→R24, R25→K25, R26→K26, R26→T26, R26→D26, A27→M27, A27→127, A27→Q27, E28→D28, E28→Q28, Y2 9→129、Y29→F29、A30→S30、A30→R30、R31→K31、R31→A31、V32→T32、V32→M32、V32→F32、Q33→N33、E34→Q34、E34→R34、E34→D34、L35→F35、L35→A35、Y36→F36、Y36→W36、K37→R37、K37→H37、K38→R38、K38→T38、C39→D39、C39→N39、R40→M40、R40→140、R40→ K40, S41→T41, S41→Q41, R42→Q42, R42→K42, R42→A42, A43→C43, A43→L43, A44→144, A44→V44, A45→H45, A45→R45, E46→R46, E 46→K46, E46→D46, V47→L47, V47→147, 148→L48, 148→F48, D49→G49, D49→S49, D49→E49, G50→A50, G50→E50, G50→K50, A51→T5 1. A51→D51、C52→A52、C52→T52、G53→S53、G53→D53、G54→S54、G54→D54、V55→L55、V55→A55、G56→S56、G56→A56、M62→L62、M62→A62、Y65→F65、Y65→G65、W66→F66、W66→H66、169→M69、169→T69、L70→V70、L70→M70、V73→A73、V73→F73、S74→E74、S74→K74.

[0100] In some aspects, the present disclosure provides a non-naturally occurring enzyme that can comprise one or more amino acid mutations in motif-1, motif-0, and thumb subunit ( Figure 2 , 3 and 4). Mutations may be made such that the encoded amino acid is modified to be polar, non-polar, basic, or acidic. Fig.16Disclosed are possible mutations of motif-0 that may improve one or more properties of the R2 enzyme from silkworm moth, wherein Fig.14The silkworm moth R2 reference sequence is disclosed in . In some cases, the following amino acid substitutions can be engineered in motif-0: Q101→N101, Q101→S101, L102→V102, L102→I102, W103→M103, W103→V103, K104→R104, K104→S104, K104→D104, P105→A105, I106→L106, I106→V106, S107→T107, S107→V107, V108→N108, V108→L108, V108→S108, E109→D109, E109→Q109, E109→L109, E110→D110, I111→V111, 1111→Mill、K112→1112、K112→R112、R115→H115、F116→L116、F116→A116、D117→C117、D117→S117、D117→E117、R119→T119、R119→N119、T120→S120、S121→A121、P122→A122、G123→A123、P124→L124、D125→N125、D125→E125、G126→S126、G126→K126、1127→M127、1127→V127、R128→T128、R128→K128、S129→L 129, S129 → H129, G130 → K130, G130 → S130, Q131 → D131, Q131 → R131, W132 → L132, W132 → A132, R133 → N133, R133 → Y133, R133 → K133, A134 → L134, A134 → M134 , V135→T135, V135→S135, P136→S136, V137→A137, V137→Q137, H138→1138, H138→A138, L139→A139, L139→M139, L139→V139, K140→R140, K140→N140, K1 40→L140, A141→deletion, E142→S142, E142→K142, E142→D142, M143→1143, M143→V143, F144→L144, F144→Y144, N145→D145, A146→L146, A146→V146, W147→F1 47. W147→L147, M148→L148, M148→V148, A149→L149, A149→F149, R150→T150, R150→H150, R150→K150, G151→R151, G151→E151, E152→R152, E152→N152,E152→D152、1153→V153、1153→C153、P154→A154、E155→K155、E155→P155、E155→Q155、E155→A155、E155→D155、1156→E156、1156→R156、1156→V156、L157→ V157, L157→F157, R158→K158, R158→L158, R158→K158, Q159→L159, Q159→M1 59. Q159→H159, Q159→N159, C160→G160, C160→S160, C160→H160, R161→K161. ,

[0101] In some aspects, the present disclosure provides a non-naturally occurring enzyme that can comprise one or more amino acid mutations in motif-1, motif-0, and thumb subunit ( Figure 2 and 3 ). Mutations can be made so that the encoded amino acid is modified to be polar, non-polar, basic or acidic. Fig.17 Possible mutations of the thumb subunit are disclosed which may improve one or more properties of the R2 enzyme from the silk moth, wherein Fig.14The silkworm moth R2 reference sequence is disclosed in . In some cases, the following amino acid substitutions can be engineered in the thumb subunit: G403→D403, G403→S403, G404→D404, G404→R404, G404→S404, K405→Q405, K405→V405, K405→R405, P406→V406, P406→Q406, P406→K406, L407→V407, L407→M407, R408→G408, R408→P408, R408→H408, R408→T408, R408→K408, Q409→A409, Q409→E409, Q409→S409, V410→M410, V410→L410, S411→D411, S411→G411, S411→K411, C412→1412, C412→H412, C 412→A412, C412→R412, V413→E413, V413→L413, V413→A413, V413→G413, E41 4→G414、E414→H414、E414→Q414、E414→K414、W416→Y416、W416→V416、W416→F416、R417→K417、R417→H417、R417→T417、R417→G417、Y418→F418、L419→V4 19. L419→1419, G420→A420, V421→1421, V421→A421, V421→H421, D422→R422, D422→W422, D422→N422, D422→YT422, D422→P422, D422→E422, F423→V423 , F423→Y423, F423→1423, E424→G424, E424→A424, E424→N424, E424→R424, E 424→T424, E424→S424, E424→D424, A425→S425, A425→H425, A425→G425, S42 6→T426, S426→A426, S426→E426, G427→A427, C428→T428, C428→P428, C428 →R428, C428→C428→M428, V429→C429, V429→1429, V429→E429, V429→A429, T 430→1430、T430→D430、T430→Q430、T430→R430、T430→T430→H430、S434→E434、S434→N434、1435→V435、1435→L435、1435→M435、S436→M436、S436→L436、S436→A436、S436→D436、S436→K436、S437→P437、S437→G437、S437→A437、S437→D437、S437→T437、A438→L438、A438→G438、A438→K438、A438→D438、A438→L438、L439→1439、L439→V439、N440→E440、N440→D440、N440→Q440、N440→K440、N441→E441、N441→R441、N441→A441、N441→Q441、1442→T442、1442→V442、S443→T443、S443→K443、S443→Q443、R444→A444、R444→C444、R444→S444、R444→Q444、R444→K444、A445→G445、A445→S445、P446→G446、L447→1447、K448→R448、P449→L449、Q450→E450、Q450→H450、Q451→E451、Q451→H451、L453→V453、L453→M453、E454→K454、E454→H454、E454→F454、E454→A454、E454→D454、1455→L455、1455→M455、1455→A455、L456→1456、R457→C457、R457→G457、R457→N457、R457→R457→K457、A458→N458、A458→T458、A458→V458、A458→S458、H459→Y459、H459→F459、H459→V459、L460→F460、L460→V460、1461→L461、1461→V461、P462→G462、R463→K463、R463→Q463、R463→G463、F464→Y464、F464→S464、F464→A464、F464→H464、Q465→T465、Q465→Y465、H466→Y466、H466→F466、G467→N467、G467→1467、G467→K467、G467→A467、F468→L468、F468→W468、V469→T469、V469→A469、V469→S469、V469→L469、L470→F470、L470→M470、L470→T470、G471→S471、G471→T471、G471→A471、N472→R472、N472→S472、N472→G472、R477→L477、R477→M477、R477→D477、R477→K477、L478→V478、L478→A478、R479→N479、R479→K479、R479→C479、R479→L479、R479→W479、M480→Q480、M480→K480、M480→T480、M480→R480、L481→G481、L481→T481、L481→M481、D482→N482、D482→E482、V483→S483、V483→K483、V483→R483、 V483→L483, Q484→A484, Q484→1484, Q484→V484, Q484→M484, 1485→T485, 1485→V485, R486→K486, R486→L486, K487→A487, K487→T487, K487→Q487, K487→G487, K487→V487, K487→R487, A488→H488, A488→T488, A488→Y488, A488→S488, G490→R490, G490→K490, Q491→R491, Q491→T491, Q491→K491.

[0102] In some aspects, the mutagenic motif-1 sequence has improved skipping activity compared to the wild-type sequence. In some aspects, the mutagenic motif-0 sequence has improved skipping activity compared to the wild-type sequence. In some aspects, the mutagenic thumb domain sequence has improved single-stranded priming efficiency compared to the wild-type sequence. In some cases, the mutagenic thumb domain sequence has improved processivity compared to the wild-type sequence. Jumping efficiency, single-stranded priming efficiency, and processivity are essential parameters for the efficiency of RNA sample conversion into DNA library.

[0103] In some cases, the host cell can be selected from, for example, bacteria, yeast, fungi, or any of a variety of other organisms that can be used as host organisms, and the modified or non-naturally occurring enzyme is produced in such organisms.

[0104] In some cases, the host is not clearly defined, and one or more enzyme activities can be incorporated into any suitable host organism using methods such as described herein. In some cases, the host is selected from bacteria, yeast, algae, cyanobacteria, fungi or plant cells or any combination thereof. Escherichia coli and Saccharomyces cerevisiae (S.cerevisiae) are particularly suitable host organisms because they are well characterized microorganisms suitable for genetic engineering. In some cases, the host is Escherichia coli.

[0105] Each enzyme described herein can be linked to other amino acid sequences, as long as the activity is functionally equivalent to the activity of the enzyme. As mentioned above, it should be understood that various enzymes or their homologues can be (multi) peptide fragments, as long as the activity is functionally equivalent to the activity of the enzyme.

[0106] In some cases, the enzyme is selected and / or engineered to exhibit high fidelity and low error rate. The fidelity of nucleotide polymerases is usually measured by error rate, i.e., the frequency of incorporating nucleotides in a manner that may violate the widely known Watson-Crick base pairing. The fidelity or error rate of a polymerase (e.g., DNA polymerase) can be measured using any suitable analysis. See, e.g., Lundburg et al., 1991 "Gene", 108: 1-6. The term "fidelity" can be used to refer to polymerization accuracy, or the ability of a polymerase to distinguish between a correct substrate and an incorrect substrate (e.g., nucleotides) when synthesizing a nucleic acid molecule (e.g., RNA or DNA) complementary to a template. The higher the fidelity of the enzyme, the fewer nucleotides the enzyme misincorporates in the growing chain during nucleic acid synthesis; that is, increasing or improving fidelity produces a more reliable polymerase with a reduced error rate (reduced misincorporation rate). In some cases, the misincorporation error rate is at most about 10-2, 10-4, 10-6, or 10-8.

[0107] In some aspects, the present disclosure relates to a non-naturally occurring or modified enzyme that is easily expressed in a functional form in a recombinant system. In some cases, the non-naturally occurring or modified enzyme is an enzyme having reverse transcriptase activity. In some cases, the non-naturally occurring or modified enzyme is a modified reverse transcriptase. In some cases, the non-naturally occurring or modified enzyme is a modified non-retroviral reverse transcriptase. In some cases, the non-naturally occurring or modified enzyme is a modified non-LTR retrotransposon. In some cases, the non-naturally occurring or modified enzyme is a modified R2 reverse transcriptase comprising a mutation in R2 motif-1 or Motif-0. In some cases, the non-naturally occurring or modified enzyme or modified polypeptide having reverse transcriptase activity can amplify the template nucleic acid molecule with a processivity of at least about 80% per base, at least 81% per base, at least 82% per base, at least 83% per base, at least 84% per base, at least about 85% per base, at least 86% per base, at least 87% per base, at least about 88% per base, at least about 89% per base, at least about 90% per base, at least about 91% per base, at least about 92% per base, at least about 93% per base, at least about 94% per base, at least about 95% per base, at least about 96% per base, at least about 97% per base, at least about 98% per base, at least about 99% per base, at least about 99.5% per base, or about 100% per base.

[0108] In some cases, the non-naturally occurring or modified enzyme or modified polypeptide having reverse transcriptase activity can or is capable of amplifying a template nucleic acid molecule with processivity measured at a temperature between about 12° C. and about 40° C. In some cases, the temperature is between about 10° C. and about 35° C., between about 12° C. and about 30° C., between about 25° C. and about 40° C., or between about 12° C. and about 42° C. In some cases, the temperature is between about 8° C. to about 50° C., between about 2° C. to about 60° C., between about 8° C. to about 42° C., between about 6° C. to about 32° C., or between about 7° C. to about 35° C.

[0109] In some cases, the non-naturally occurring or modified enzyme or modified polypeptide having reverse transcriptase activity can or is capable of amplifying a template nucleic acid molecule with a processivity of at least about 80% per base at a temperature of about or at most about 4°C, about or at most about 8°C, about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, or about or at most about 42°C; at least about 85% per base at a temperature of about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, or about or at most about 42°C. at least about 89% processivity per base at a temperature of about or at most about 4°C, about or at most about 8°C, about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, about or at most about 42°C, about or at most about 45°C, about or at most about 50°C; at least about 90% processivity per base at a temperature of about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, or about or at most about 42°C; ℃, about or at most about 40℃, about or at most about 42℃, about or at most about 45℃, about or at most about 50℃; at least about 91% processivity per base at a temperature of about or at most about 4℃, about or at most about 8℃, about or at most about 12℃, about or at most about 15℃, about or at most about 20℃, about or at most about 25℃, about or at most about 30℃, about or at most about 35℃, about or at most about 40℃ or about or at most about 42℃; at least about 85% per base at a temperature of about or at most about 12℃, about or at most about 15℃, about or at most about 20℃, about or at most about 25℃, about or at most about 30℃, about or at most about 35℃, about or at most about 40℃, about or at most about 42℃ , about or at most about 45°C, about or at most about 50°C; at least about 95% per base at a temperature of about or at most about 4°C, about or at most about 8°C, about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, or about or at most about 42°C; at least about 85% processivity per base at a temperature of about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, about or at most about 42°C, about or at most about 45°C, about or at most about 50°C;At least about 99% processivity per base at a temperature of about or at most about 4°C, about or at most about 8°C, about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, or about or at most about 42°C; at least about 85% processivity per base at a temperature of about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C , about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, about or at most about 42°C, about or at most about 45°C, about or at most about 50°C; at least about 99.5% processivity per base at a temperature of about or at most about 4°C, about or at most about 8°C, about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, or about or at most about 42°C; At least about 85% processivity per base at a temperature of about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, about or at most about 42°C, about or at most about 45°C, about or at most about 50°C; or about 100% processivity per base at a temperature of about or at most about 4°C, about or at most about 8°C, about or at most about 12°C, about or at most about 15°C , about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, or about or at most about 42°C; at least about 85% processivity per base at a temperature of about or at most about 12°C, about or at most about 15°C, about or at most about 20°C, about or at most about 25°C, about or at most about 30°C, about or at most about 35°C, about or at most about 40°C, about or at most about 42°C, about or at most about 45°C, or about or at most about 50°C. ;

[0110] In some cases, the non-naturally occurring or modified enzyme or modified polypeptide having reverse transcriptase activity can or is capable of amplifying a template nucleic acid molecule with the following processivity at a temperature of at least about 80% processivity at a temperature of up to about 35°C; at least about 85% processivity per base at a temperature of up to about 40°C; at least about 88% processivity per base at a temperature of up to about 35°C; at least about 89% processivity per base at a temperature of up to about 40°C; at least about 90% processivity per base at a temperature of up to about 35°C; at least about 91% processivity per base at a temperature of up to about 35°C; at least about 92% processivity per base at a temperature of up to about 40°C. ℃; at least about 93% processivity per base at a temperature of up to about 35℃; at least about 94% processivity per base at a temperature of up to about 40℃; at least about 95% processivity per base at a temperature of up to about 35℃; at least about 96% processivity per base at a temperature of up to about 40℃; at least about 97% processivity per base at a temperature of up to about 35℃; at least about 98% processivity per base at a temperature of up to about 40℃; at least about 99% processivity per base at a temperature of up to about 40℃; at least about 99.5% processivity per base at a temperature of up to about 40℃; or about 100% processivity per base at a temperature of up to about 40℃.

[0111] In some cases, the improved enzyme property is selected from at least one of improved stability (e.g., improved thermostability), improved specific activity, improved protein expression, improved purification, improved processivity, improved strand displacement, improved template hopping, improved DNA / RNA affinity, improved single-stranded DNA priming, and improved fidelity. In some cases, the non-naturally occurring enzyme or modified enzyme or modified polypeptide having reverse transcriptase activity amplifies the template nucleic acid molecule. In some cases, the non-naturally occurring enzyme or modified enzyme or modified polypeptide having reverse transcriptase activity that amplifies the template nucleic acid molecule has a performance index greater than about 1, greater than about 2, greater than about 3, greater than about 4, greater than about 5, greater than about 6, greater than about 7, greater than about 8, greater than about 9, greater than about 10, greater than about 15, greater than about 20, greater than about 25, greater than about 30, greater than about 35, greater than about 40, greater than about 45, greater than about 50, greater than about 60, greater than about 70, greater than about 80, greater than about 90, or greater than about 100 for at least one enzyme property. In some cases, the enzyme properties and / or performance indicators are achieved at a temperature of at least about 50°C, at least about 42°C, at least about 40°C, at least about 39°C, at least about 38°C, at least about 37°C, at least about 36°C, at least about 35°C, at least about 34°C, at least about 33°C, at least about 32°C, at least about 31°C, or at most about 32°C. , equal to or less than or at most about 30°C, equal to or less than or at most about 29°C, equal to or less than or at most about 28°C, equal to or less than or at most about 27°C, equal to or less than or at most about 26°C, equal to or less than or at most about 25°C, equal to or less than or at most about 23°C, equal to or less than or at most about 20°C, equal to or less than or at most about 15°C, equal to or less than or at most about 13°C, equal to or less than or at most about 12°C, equal to or less than or at most about 10°C, equal to or less than or at most about 8°C, equal to or less than or at most about 4°C.In some cases, the non-naturally occurring enzyme or modified enzyme (e.g., a modified reverse transcriptase) or modified polypeptide with reverse transcriptase activity exhibits a processivity for a given nucleotide substrate that is higher than the processivity of a reference enzyme or reference polypeptide for the same nucleotide substrate by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 37.5%, at least about 40%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 100%, In some cases, the non-naturally occurring enzyme is a non-naturally occurring reverse transcriptase. In some cases, the modified enzyme is a modified reverse transcriptase.

[0112] The present disclosure relates to a process and / or method requiring significantly less hands-on time, for example, compared to other methods for RNA sequencing and / or liquid biopsy, the execution of the scheme is much simpler and the duration required is much shorter. In some cases, the method and process of the present disclosure include a scheme less than about 2 hours and / or a hands-on time less than about 30 minutes. In some cases, the scheme is less than about 20 hours, less than about 15 hours, less than about 12 hours, less than about 11 hours, less than about 10 hours, less than about 9 hours, less than about 8 hours, less than about 7 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour or less than about 30 minutes. In some cases, the hands-on time is less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 50 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, or less than about 15 minutes.

[0113] In some aspects, the present disclosure provides a method for simultaneously amplifying ribonucleic acid (RNA) molecules and deoxyribonucleic acid (DNA) molecules. In some cases, the method includes providing a reaction mixture comprising the mRNA, DNA, and a non-naturally occurring enzyme, each of which includes (i) palm and finger domains derived from an R2 retrotransposon; (ii) a thumb domain derived from an R2 retrotransposon; and (iii) an endonuclease domain derived from an R2 retrotransposon. In some cases, the method includes subjecting the reaction mixture to conditions sufficient to amplify the RNA and DNA, thereby obtaining an amplified product of the RNA and the DNA. In some cases, the DNA is a complementary DNA derived from a subset of the mRNA in the reaction mixture.

[0114] In some cases, the non-naturally occurring or modified enzyme (e.g., a non-naturally occurring or modified reverse transcriptase, a non-naturally occurring or modified non-LTR retrotransposon, a non-naturally occurring or modified R2 reverse transcriptase) or a modified polypeptide having reverse transcriptase activity exhibits a misincorporation error rate of about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 10% or less, about 15% or less, about 20% or less, about 2 ... 15%, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4%, about 3% or less, about 2%, about 1% or less, about 0.01%, about 0.001%, about 0.0001%, about 0.00001%, about 0.000001%, or about 0.0000001%.

[0115] In some cases, the non-naturally occurring or modified enzyme (e.g., a non-naturally occurring or modified reverse transcriptase, a non-naturally occurring or modified non-LTR retrotransposon, a non-naturally occurring or modified R2 reverse transcriptase) or a modified polypeptide having reverse transcriptase activity produces one or more nucleic acid (e.g., cDNA) molecules that are complementary to a template with an error rate that is at least about 10,000 times lower, at least about 1,500 times lower, at least about 1,000 times lower, at least about 500 times lower, at least about 100 times lower, or at least about 150 times lower than an unmodified or naturally occurring enzyme or an unmodified polypeptide having reverse transcriptase activity. At least about 95 times lower, at least about 90 times lower, at least about 85 times lower, at least about 80 times lower, at least about 75 times lower, at least about 70 times lower, at least about 65 times lower, at least about 60 times lower, at least about 55 times lower, at least about 50 times lower, at least about 45 times lower, at least about 40 times lower, at least about 35 times lower, at least about 30 times lower, at least about 25 times lower, at least about 20 times lower, at least about 15 times lower, at least about 10 times lower, at least about 9 times lower, at least about 8 times lower, at least about 7 times lower, at least about 6 times lower, at least about 5 times lower, at least about 4 times lower, at least about 3 times lower, at least about 2 times lower, or at least about 1 times lower.

[0116] In some cases, the sequencing error rate will be equal to or less than about 1 / 100,000 bases. In some cases, the error rate of nucleotide sequence determination is equal to or less than about 1 / 10 bases, 1 / 20 bases, 3 / 100 bases, 1 / 100 bases, 1 / 1000 bases, and 1 / 10,000 bases.

[0117] In some cases, a modified enzyme (e.g., a modified reverse transcriptase), a modified reverse transcriptase, a non-naturally occurring enzyme, a modified polypeptide with reverse transcriptase activity comprises at least one modification relative to a wild-type, unmodified counterpart, or a naturally occurring enzyme. In some cases, a modified non-LTR retrotransposon comprises at least one modification of a wild-type or unmodified non-LTR retrotransposon. In some cases, a modified R2 reverse transcriptase comprises at least one modification of a wild-type or unmodified R2 reverse transcriptase. In some cases, a modified reverse transcriptase comprises at least one modification of a wild-type or unmodified reverse transcriptase. In some cases, a modified polypeptide with reverse transcriptase activity comprises at least one modification of a wild-type or unmodified polypeptide with reverse transcriptase activity. In some cases, the modification comprises at least one truncation (e.g., N-terminal truncation, C-terminal truncation, and / or N-terminal and C-terminal truncation). In some cases, the modification comprises site-specific incorporation and / or amino acid addition and / or deletion and / or substitution at the position of interest. In some cases, modification enhances the biological properties of modified enzyme or modified polypeptide relative to wild-type or unmodified enzyme or polypeptide. In some cases, modification improves at least one enzyme property of modified enzyme or polypeptide relative to wild-type or unmodified enzyme or polypeptide. In some cases, modification is used as the connection point of for example labeling and protein half-life extender and in order to attach variant to the surface of solid support. In some cases, the disclosure relates to the method for producing the cell that can produce modified enzyme (for example modified reverse transcriptase) or modified polypeptide, and the method for producing the carrier containing DNA or RNA encoding modified enzyme (for example modified reverse transcriptase) or modified polypeptide. In some cases, truncation is based on two-step method. In some cases, the first step for selecting truncation includes analyzing the domain and motif structure and function of a class of enzyme or protein or polypeptide. In some cases, enzyme or protein or polypeptide is non-LTR retrotransposon, reverse transcriptase, R2 reverse transcriptase, LTR retrotransposon, R2 non-LTR retrotransposon or its any combination. In some cases, enzymes or proteins or polypeptides are from different organisms. In some cases, all domains of enzymes or proteins or polypeptides are present. In some cases, all domains are present to ensure reverse transcriptase activity. In some cases, all domains are present to ensure the unique properties necessary for the disclosure. In some cases, the domain responsible for reverse transcriptase activity is not modified. In some cases, the R2 domain does not include modifications. In some cases, the R2 domain may include modifications. In some cases, truncated variants display expression levels. In some cases, the truncated variants that display promising expression levels are further subjected to minor adjustments of sequence (step two). In some cases, minor adjustments of sequence include amino acid deletions, insertions and / or substitutions. In some cases, amino acid deletions, insertions and / or substitutions may include one or more amino acids.In some cases, amino acid deletions, insertions and / or substitutions further optimize expression and / or stability (eg, thermal stability).

[0118] In some cases, the modified enzyme (e.g., a modified reverse transcriptase), the modified reverse transcriptase, or the modified polypeptide has an N-terminal truncation, a C-terminal truncation, or both relative to a wild-type or unmodified enzyme (e.g., a wild-type reverse transcriptase) or a wild-type or unmodified polypeptide. In some cases, the polymerase comprises an N-terminal truncation, a C-terminal truncation, or both. In some cases, the modified reverse transcriptase comprises an N-terminal truncation, a C-terminal truncation, or a combination of an N-terminal truncation and a C-terminal truncation. In some cases, the modified enzyme comprises an N-terminal truncation, a C-terminal truncation, or a combination of an N-terminal truncation and a C-terminal truncation. In some cases, the modified polypeptide comprises an N-terminal truncation, a C-terminal truncation, or a combination of an N-terminal truncation and a C-terminal truncation. In some cases, the modified reverse transcriptase, the modified enzyme, the modified polypeptide, the modified non-LTR retrotransposon, or the modified R2 reverse transcriptase comprises a truncation of less than about 100 amino acid residues. In some cases, the modified reverse transcriptase, modified enzyme, modified polypeptide, modified non-LTR retrotransposon, or modified R2 reverse transcriptase comprises at least one of: (a) an amino-terminal truncation of less than about 400 amino acid residues and (b) a carboxyl-terminal truncation of less than about 400 amino acid residues. In some cases, the modified reverse transcriptase, modified enzyme, modified polypeptide, modified non-LTR retrotransposon, or modified R2 reverse transcriptase lacks up to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or both of the N-terminus, the C-terminus, or both. , about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 50, about 75, about 100, about 120, about 150, about 175, about 200, about 220, about 250, about 275, about 280, about 290, about 300, about 325, about 350, about 375, about 380, about 390, about 400, or about 450 amino acids.In some cases, the modified reverse transcriptase, modified enzyme, modified polypeptide, modified non-LTR retrotransposon, or modified R2 reverse transcriptase may alternatively or additionally have up to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 In some embodiments, the invention relates to a polypeptide having an internal deletion of about 21, about 22, about 23, about 24, or about 25 amino acids, about 30, about 50, about 75, about 100, about 120, about 150, about 175, about 200, about 220, about 250, about 275, about 280, about 290, about 300, about 325, about 350, about 375, about 380, about 390, or about 450 amino acids in total. In some cases, the N-terminal truncation, the C-terminal truncation, or both can comprise a deletion of about 1 to about 50 amino acids, about 1 to about 25, about 1 to about 70, about 10 to about 50, about 20 to about 30, about 15 to about 100, about 1 to about 150, about 15 to about 60, about 15 to about 40, about 1 to about 10, about 10 to 35, about 50 to about 100, about 20 to about 150, about 200 to about 350, about 25 to about 350, about 150 to about 400, about 50 to about 400, about 50 to about 450, about 200 to about 400, or about 50 to about 350, or about 50 to about 400 amino acids. In some cases, the N-terminal truncation removes at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 50, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 90, at least about 95, at least about 100, at least about 120, at least about 130, at least about 140, at least about 150, at least about 175, at least about 200, at least about 220, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 375, or at least about 400 amino acids.In some cases, the C-terminal truncation removes at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 50, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 90, at least about 95, at least about 100, at least about 120, at least about 130, at least about 140, at least about 150, at least about 175, at least about 200, at least about 220, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 375, or at least about 400 amino acids. In some cases, the N-terminal truncation lacks about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 65, about 70, about 75, about 80, about 90, about 95, about 100, about 120, about 130, about 140, about 150, about 175, about 200, about 220, about 250, about 275, about 300, about 325, about 350, about 375, or about 400 amino acids. In some cases, the C-terminal truncation lacks about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 65, about 70, about 75, about 80, about 90, about 95, about 100, about 120, about 130, about 140, about 150, about 175, about 200, about 220, about 250, about 275, about 300, about 325, about 350, about 375, or about 400 amino acids. In some cases, the N-terminal truncation lacks at most about 5, at most about 10, at most about 15, at most about 20, at most about 25, at most about 30, at most about 35, at most about 40, at most about 50, at most about 60, at most about 65, at most about 70, at most about 75, at most about 80, at most about 90, at most about 95, at most about 100, at most about 120, at most about 130, at most about 140, at most about 150, at most about 175, at most about 200, at most about 220, at most about 250, at most about 275, at most about 300, at most about 325, at most about 350, at most about 375, or at most about 400 amino acids.In some cases, the C-terminal truncation lacks at most about 5, at most about 10, at most about 15, at most about 20, at most about 25, at most about 30, at most about 35, at most about 40, at most about 50, at most about 60, at most about 65, at most about 70, at most about 75, at most about 80, at most about 90, at most about 95, at most about 100, at most about 120, at most about 130, at most about 140, at most about 150, at most about 175, at most about 200, at most about 220, at most about 250, at most about 275, at most about 300, at most about 325, at most about 350, at most about 375, or at most about 400 amino acids. In some cases, the truncation comprises an N-terminal truncation that removes at least about, at most about, or about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 amino acids. In some cases, the truncation includes a C-terminal truncation that removes at least about, at most about, or about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 amino acids. In some cases, the N-terminal truncation, the C-terminal truncation, or both can be more than about 500 amino acids, more than about 1000 amino acids, more than about 1500 amino acids, more than about 2000 amino acids, more than about 5000 amino acids, more than about 10000 amino acids, more than about 100000 amino acids, more than about 1000000 amino acids.

[0119] In some cases, the truncation of the region that affects the functional activity of the protein or enzyme can be engineered. In some cases, the truncation of the region that does not affect the functional activity of the protein or enzyme can be engineered. The truncation can include less than about 5, less than about 10, less than about 15, less than about 20, less than about 25, less than about 30, less than about 35, less than about 40, less than about 45, less than about 50, less than about 60, less than about 70, less than about 80, less than about 90, less than about 100, less than about 125, less than about 150, less than about 200, less than about 250, less than about 300, less than about 350, less than about 400 or more amino acid truncation. The truncations may comprise truncations of more than about 5, more than about 10, more than about 15, more than about 20, more than about 25, more than about 30, more than about 35, more than about 40, more than about 45, more than about 50, more than about 60, more than about 70, more than about 80, more than about 90, more than about 100, more than about 125, more than about 150, more than about 200, more than about 250, more than about 300, more than about 350, more than about 400, or more amino acids. The truncation can comprise about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 87%, about 90%, about 92%, about 95%, or about 100% truncation of the polypeptide or enzyme.

[0120] In some cases, the variant or modified enzyme or modified protein can comprise one or more modifications at an amino acid position. In some cases, the variant, mutant or modified polypeptide or enzyme of the present disclosure can have an increased activity compared to a corresponding unmutated or unmodified or wild-type polymerase or compared to one or more polymerases (e.g., RNA-dependent DNA polymerases or reverse transcriptases), such as increased RNA-dependent DNA polymerase activity or DNA-dependent DNA polymerase activity. In some cases, the polymerase or reverse transcriptase with increased activity can be a modified polymerase or modified reverse transcriptase having at least about 5% increase, at least about 10% increase, at least about 25% increase, at least about 30% increase, at least about 50% increase, at least about 100% increase, at least about 150% increase, at least about 200% increase, at least about 300% increase, at least about 500% increase, at least about 1,000% increase, at least about 2,500% increase, or at least about 5,000% increase compared to: (1) the corresponding unmutated or wild-type enzyme; or (2) a specific polymerase (e.g., RNA-dependent DNA polymerase, reverse transcriptase) or a specific reverse transcriptase or a group of polymerases or a group of reverse transcriptases. In some cases, the activity of the modified polymerase or modified reverse transcriptase of the present disclosure can be increased by about 5% to about 5,000%, about 5% to about 2,500%, about 5% to about 1000%, about 5% to about 500%, about 5% to about 250%, about 5% to about 100%, about 5% to about 50%, about 5% to about 25%, about 25% to about 5,000%, about 25% to about 2,500%, about 25% to about 1,000%, about 25% to about 500%, about 25% to about 250%, about 25% to about 100%, about 100% to about 5,000%, about 100% to about 2,500%, about 100% to about 1000%, about 100% to about 500%, or about 100% to about 250%. The increase in RNA-dependent DNA polymerase activity and / or DNA-dependent DNA polymerase activity of the modified polymerases or modified reverse transcriptases of the present disclosure can also be measured based on the relative activity compared to: (1) the corresponding unmodified or wild-type enzyme; or (2) a specific polymerase (e.g., RNA-dependent DNA polymerase, reverse transcriptase) or a specific reverse transcriptase or a group of polymerases or a group of reverse transcriptases.In some cases, the increase in relative activity of a modified polymerase or modified reverse transcriptase of the present disclosure is at least about 1.1, 1.2, 1.5, 2, 5, 10, 25, 50, 75, 100, 150, 200, 300, 500, 1,000, 2,500, 5,000, 10,000, or 25,000-fold when compared to: (1) a corresponding unmodified or wild-type enzyme; or (2) a specific polymerase (e.g., RNA-dependent DNA polymerase, reverse transcriptase) or a specific reverse transcriptase or a group of polymerases or a group of reverse transcriptases. Thus, the modified polymerase or modified reverse transcriptase of the present disclosure may have an increased RNA-dependent DNA polymerase and / or increased DNA-dependent DNA polymerase activity of about 1.1 times to about 25,000 times, about 1.1 times to about 10,000 times, about 1.1 times to about 5,000 times, about 1.1 times to about 2,500 times, about 1.1 times to about 1,000 times, about 1.1 times to about 500 times. , about 1.1 times to about 250 times, about 1.1 times to about 50 times, about 1.1 times to about 25 times, about 1.1 times to about 10 times, about 1.1 times to about 5 times, about 5 times to about 25,000 times, about 5 times to about 5,000 times, about 5 times to about 1,000 times, about 5 times to about 500 times, about 5 times to about 100 times, about 5 times to about 50 times, about 5 times to about 25 times, about 50 times to about 25,000 times, about 50 times to about 5,000 times, about 50 times to about 1,000 times, about 50 times to about 500 times, about 50 times to about 100 times, about 100 times to about 25,000 times, about 1,000 times to about 25,000 times, about 4,000 times to about 25,000 times, about 10,000 times to about 25,000 times, about 15,000 times to about 25,000 times, about 1,000 times to about 10,000 times, about 2,500 times to about 25,000 times. times, to about 10,000 times, about 5,000 times to about 10,000 times, about 7,500 times to about 10,000 times, about 1,000 times to about 15,000 times, about 2,500 times, to about 15,000 times, about 5,000 times to about 15,000 times, about 7,500 times to about 15,000 times, about 10,000 times to about 15,000 times, or about 12,500 times to about 15,000 times.

[0121] In some cases, the polypeptide, protein, enzyme, modified enzyme (e.g., modified reverse transcriptase), modified polypeptide, non-naturally occurring enzyme or variant comprises a fusion with, but not limited to, a protein, a domain, a fusion partner, a carrier protein, a target sequence, an antigenic determinant, or any combination thereof. In some cases, a reverse transcriptase or a modified reverse transcriptase is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof. In some cases, a non-LTR retrotransposon or a modified non-LTR retrotransposon is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof. In some cases, a modified LTR retrotransposon is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof. In some cases, a modified R2 non-LTR retrotransposon is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof. In some cases, a modified R2 reverse transcriptase is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof. In some cases, the modified reverse transcriptase is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof. In some cases, the variant is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof. In some cases, the polypeptide having reverse transcriptase activity is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof.

[0122] In some cases, the fused polypeptide, protein, enzyme, modified enzyme (e.g., modified reverse transcriptase), modified polypeptide, non-naturally occurring enzyme, or variant thereof, has increased stability (e.g., increased thermostability), increased shelf life, increased active moiety, and / or improved purification compared to a wild-type counterpart, a naturally occurring enzyme, or an unfused polypeptide, protein, enzyme, or variant thereof. In some cases, the modified reverse transcriptase comprises a fusion partner or a carrier protein. In some cases, the selection of a fusion protein, domain, fusion partner, target sequence, antigenic determinant, or any combination thereof is based on a mechanism that causes decreased or increased stability (e.g., increased thermostability), decreased or increased shelf life, and / or decreased or increased expression levels (Costa et al., Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system Front Microbiol. 2014 Feb 19;5:63). In some cases, the fusion tag improves the solubility of its partner protein. In some cases, the fusion protein forms a micelle-like structure. In some cases, the micelle-like structure is a misfolded or unfolded protein that is shielded and protected from the influence of the solvent and / or the soluble protein domain is outward. In some cases, the fusion partner attracts a chaperone. In some cases, the fusion tag forces its partner protein to enter the folding pathway mediated by the chaperone. In some cases, MBP and / or N utilization substance (NusA) are two fusion tags that present this mechanism. In some cases, the fusion partner has an inherent chaperone protein-like activity. In some cases, the hydrophobic small piece of the fusion tag interacts with the partially folded chaperone protein (passenger protein), thereby preventing self-aggregation and promoting proper folding. In some cases, the solubility enhancement partner may play a passive role in the folding of its target protein, thereby reducing the chance of protein aggregation. In some cases, the fusion partner nets the charge. In some cases, the strongly acidic fusion partner inhibits protein aggregation. In some cases, the fusion is with, but is not limited to, Fh8, MBP, NusA, Trx, SUMO, GST, SET, GB1, ZZ, HaloTag, SNUT, Skp, T7PK, EspA, Mocr, Ecotin, CaBP, ArsC, IF2 domain I, an expression tag, an expression tag that is part of IF2 domain I, RpoA, SlyD, Tsf, RpoS, PotD, Crr, msyB, yjgD, rpoD, His6, or any combination thereof.In some cases, the fusion enhances protein solubility and / or purification. In some cases, Fh8 can be used as an effective solubility enhancement and / or stable purification partner. In some cases, the Fh8 fusion tag has an amino acid sequence comprising MPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGK L DLKELVSILSS (SEQ ID NO: 21). In some cases, the codon optimized sequence comprises.

[0123] ATGCCGTCTGTTCAGGAAGTTGAAAAACTGCTGCACGTTCTGGACCGTAACGGTGA

[0124] CGGTAAAGTTTCTGCGGAAGAACTGAAAGCGTTCGCGGACGACTCTAAATGCCCGC

[0125] TGGACTCTAACAAAATCAAAGCGTTCATCAAAGAACACGACAAAAACAAAGACGGTAAACTGGACCTGAAAGAACTGGTTTCTATCCTGTCTTCTTAG (SEQ ID NO: 22). In some cases, the enzyme or modified enzyme (e.g., modified reverse transcriptase), or protein (e.g., modified protein, or polypeptide (e.g., modified polypeptide), or variant, or product, or nucleic acid molecule, or cDNA molecule, or template, or receptor nucleic acid molecule, or primer, or RNA, or DNA, or fragmented nucleic acid, or degraded nucleic acid of the present disclosure can comprise one or more tags. In some cases, the fragmented or degraded RNA or DNA or variants thereof can comprise one or more tags. In some cases, the R2 reverse transcriptase or variant thereof can comprise one or more tags. In some cases, the non-LTR retrotransposon protein or having reverse transcriptase activity The polypeptide or variant thereof may comprise one or more tags. In some cases, the cDNA molecule may comprise one or more tags. In some cases, the tag may be captured on a solid support, thereby facilitating the separation of the enzyme or protein or polypeptide or variant or product of the present disclosure. In some cases, the tag may be biotin that can be recognized by avidin. The affinity tag may comprise multiple biotin residues for increasing binding to multiple avidin molecules. In some cases, the tag may comprise a functional group, such as an azido or acetylene group, which enables capture by copper (I)-mediated click chemistry (see HC Kolb and KB Sharpless, Drug Discovery Today). Discovery Today, 2003, 8(24), 1128-1137). In some cases, the tag may include an antigen that can be captured by an antibody bound to a solid support. In some cases, the tag may include, but is not limited to, a His tag, a His6 tag, a calmodulin tag, CBP, CYD (covalent but detachable NorpD peptide), Strep II, a FLAG tag, an HA tag, a Myc tag, an S tag, an SBP tag, Softag-1, Softag-3, a V5 tag, an Xpress tag, an Isopeptag, a SpyTag, a B, an HPC (protein C heavy chain) peptide tag, a GST, an MBP, a biotin, a biotin carboxyl carrier protein, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose binding protein tag, a Nus tag, a Strep tag, a thioredoxin tag, and combinations thereof. In some cases, the tagged molecules may be sequenced.

[0126] In some cases, the molecular barcode can be connected to any region of the molecule. For example, the molecular barcode can be connected to the 5' end or 3' end of a polynucleotide (e.g., DNA, RNA). For example, the target-specific region of the molecular barcode includes a sequence complementary to the sequence in the 5' region of the molecule. The target-specific region of the molecular barcode can also include a sequence complementary to the sequence in the 3' region of the molecule. In some cases, the molecular barcode connects to regions within a gene or a gene product. For example, genomic DNA is fragmented, and a sample tag or a molecular identification marker is connected to fragmented DNA. In other cases, RNA molecules undergo alternative splicing, and the molecular barcode is connected to a variant of alternative splicing. In another example, the polynucleotide is digested, and the molecular barcode is connected to the digested polynucleotide. In another example, the target-specific region of the molecular barcode includes a sequence complementary to the sequence within the molecule.

[0127] In some cases, the methods of the present disclosure include introducing a biotin moiety or another affinity purification moiety into, for example, a nucleic acid molecule, such as DNA, RNA, or a combination of DNA and RNA. In some cases, the method further includes immobilizing the nucleic acid molecule with the affinity purification tag on a solid support. In some cases, the solid support is an agarose resin or magnetic beads combined with an affinity purification substance, such as avidin, streptavidin, chitin, glutathione, etc.

[0128] In some cases, enzymes or proteins or polypeptides or variants or products of the present disclosure can be bound to solid supports. In some cases, nucleic acids (such as RNA or DNA) or variants thereof of fragmentation or degradation can be bound to solid supports. In some cases, R2 reverse transcriptase or its variants can be bound to solid supports. In some cases, non-LTR retrotransposon proteins or polypeptides or their variants with reverse transcriptase activity can be bound to solid supports. In some cases, cDNA molecules can be bound to solid supports. In some cases, solid supports can be glass, plastics, porcelain products, resins, agarose, silicon dioxide or other materials. In some cases, solid supports can be substrates, gels, microbeads, magnetic beads, membranes or other suitable shapes and sizes that are substantially flat. In some cases, microbeads can have a diameter between 10nm and several millimeters. In some cases, solid supports can be non-porous or porous, with various pore densities and pore sizes. In some cases, DNA and / or RNA fragments can be captured on solid supports, and unwanted DNA and / or RNA can be washed off. In some cases, DNA and / or RNA fragments can be released from a solid support, for example, by using restriction enzymes.

[0129] In some cases, the solid carrier may include a target nucleic acid binding region, wherein the target nucleic acid binding region includes a sequence selected from the group consisting of a gene-specific sequence, an oligo-dT sequence, a random polymer, and any combination thereof. In some cases, the solid carrier further includes a target nucleic acid or its complement. In some cases, the solid carrier includes a plurality of target nucleic acids or its complement, comprising about 0.01% to about 100% of the transcript of the transcriptome of an organism or its complement, or about 0.01% to about 100% of the gene of the genome of an organism or its complement. In some cases, the cell markers of a plurality of oligonucleotides include a first random sequence connected to a second random sequence by a first marker connecting sequence; and the molecular markers of a plurality of oligonucleotides include a random sequence. In some cases, the solid carrier is selected from the group consisting of: polydimethylsiloxane (PDMS) solid carrier, polystyrene solid carrier, glass solid carrier, polypropylene solid carrier, agarose solid carrier, gelatin solid carrier, magnetic solid carrier, pluronic (pluronic) solid carrier, and any combination thereof. In some cases, a plurality of oligonucleotides include a linker, the linker includes a linker functional group, and the solid support includes a solid support functional group; wherein the solid support functional group and the linker functional group are connected to each other. In some cases, the linker functional group and the solid support functional group are individually selected from the group consisting of: C6, biotin, streptavidin, primary amine, aldehyde, ketone and any combination thereof. In some cases, the molecular labeling of a plurality of oligonucleotides includes at least 15 nucleotides.

[0130] In some cases, the fusion partner can be removed from its target protein by enzymatic cleavage, chemical cleavage and / or by using an in vivo cleavage strategy. In some cases, a protease can be used to remove the tag. In some cases, the protease can be an endoprotease, a serine protease, a factor Xa, an enterokinase, an α-thrombin, a viral protease, a tobacco etch virus (TEV), a human rhinovirus 3C protease, a SUMO protease, an exoprotease, a metallocarboxypeptidase or an aminopeptidase. In some cases, the fusion tag can be removed by two purification steps. In some cases, the initial affinity purification step includes (for example, by a histidine tag at the N-terminus of the fusion protein) to cleave the purified fusion protein mixed into a solution with an endoprotease (for example, a his-tagged protease). The cracked target protein can be recovered in a flow sample after the second affinity purification step, wherein the cracked fusion tag and the added protease are collected in the sample through the elution.

[0131] In some cases, the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity without thermal cycling, is capable of template hopping, and / or produces nucleic acid molecules (e.g., cDNA molecules). In some cases, the modified reverse transcriptase, modified enzyme, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity at a temperature in the following range, is capable of template hopping and / or produces nucleic acid cDNA molecules: about 25°C to about 42°C, about 12°C to about 42°C, about 8°C to about 50°C, about 4°C to about 60°C, about 27°C to about 35°C, about 28°C to about 33°C, about 29°C to about 32°C, about 30°C to about 37°C, about 26°C to about 38°C, about 30°C to about 37°C, about 25°C to about 32°C, about 29°C to about 31°C, about 27°C to about 38°C, about 29°C to about 38°C. In some cases, the non-naturally occurring enzyme, modified reverse transcriptase, modified enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity at about 30°C, or at about 35°C, or at about 25°C, capable of template hopping and / or producing nucleic acid molecules (e.g., cDNA molecules). In some cases, the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity at temperatures of about 38°C or less, about 42°C or less, about 50°C or less, about 60°C or less, about 35°C or less, about 30°C or less, about 28°C or less, about 25°C or less, about 20°C or less, about 12°C or less, about 8°C or less, or about 4°C or less. In some cases, the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity at a temperature of about 36° C. or less, is capable of template hopping, and / or produces nucleic acid molecules (e.g., cDNA molecules). In some cases, the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity at room temperature, is capable of template hopping, and / or produces nucleic acid molecules (e.g., cDNA molecules).In some cases, the modified enzymes, modified reverse transcriptases, non-naturally occurring enzymes, or modified polypeptides having reverse transcriptase activity of the present disclosure exhibit activity, are capable of template jumping, and / or produce nucleic acid molecules (e.g., cDNA molecules) at temperatures of about or up to about 8°C, about or up to about 12°C, about or up to about 20°C, about or up to about 25°C, about or up to about 28°C, about or up to about 30°C, about or up to about 31°C, about or up to about 32°C, about or up to about 33°C, about or up to about 34°C, about or up to about 35°C, about or up to about 36°C, about or up to about 39°C, about or up to about 40°C, about or up to about 41°C, about or up to about 42°C, about or up to about 50°C, about or up to about 55°C, about or up to about 60°C. In some cases, the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity, is capable of template hopping, and / or produces nucleic acid molecules (e.g., cDNA molecules) at a temperature equal to or less than about any of the following temperatures: between about 42°C and about 80°C, or between about 35°C and about 80°C, or between about 30°C and about 50°C, or between about 8°C and about 50°C, or between about 12°C and about 42°C.

[0132] In some cases, the modified enzyme, modified reverse transcriptase, modified polypeptide having reverse transcriptase activity, or non-naturally occurring enzyme of the present disclosure has at least one altered characteristic relative to an unmodified or naturally occurring enzyme. In some cases, the altered characteristic enables the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity to generate nucleic acid molecules and / or complementary deoxyribonucleic acid (cDNA) molecules from a template nucleic acid molecule without thermal cycling. In some cases, the modified enzyme, modified reverse transcriptase, modified polypeptide having reverse transcriptase activity, or non-naturally occurring enzyme of the present disclosure is capable of producing one or more copies of a nucleic acid molecule or cDNA molecule with an error rate of at most about 0.5%, at most about 1%, at most about 1.5%, at most about 2%, at most about 2.5%, at most about 3%, at most about 3.5%, at most about 4%, at most about 4.5%, at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 40%, at most about 45%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, or at most about 80%. In some cases, the modified enzyme, modified reverse transcriptase, modified polypeptide with reverse transcriptase activity or non-natural enzyme of the present disclosure is a variant of any one of the sequences disclosed herein. In some cases, the modified enzyme, modified reverse transcriptase, modified polypeptide with reverse transcriptase activity or non-natural enzyme of the present disclosure is a variant of any one of the sequences provided in SEQ ID No: 1-20. In some cases, the modified enzyme, modified reverse transcriptase, modified polypeptide with reverse transcriptase activity or non-natural enzyme of the present disclosure has at least one change feature of improved enzyme properties relative to unmodified or naturally occurring enzymes. In some cases, the change feature of at least one improved enzyme property includes at least one of the following: improved / improved stability (e.g., improved / improved thermal stability), increased / improved specific activity, increased / improved protein expression, increased / improved purification, increased / improved processivity, increased / improved chain displacement, increased / improved template hopping, improved single-stranded DNA priming and increased / improved fidelity.

[0133] In one embodiment, the present disclosure relates to a non-naturally occurring enzyme that reverse transcribes a template nucleic acid molecule to produce a complementary deoxyribonucleic acid (cDNA) product and amplifies the cDNA product with a processivity of at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% per base measured at about 12°C, about 15°C, about 20°C, about 25°C, about 30°C, about 32°C, about 35°C, about 40°C.

[0134] In some cases, the non-naturally occurring enzyme has a performance index greater than about 1.0 for at least one enzyme property. In some cases, the enzyme property is at least one of the group consisting of improved stability (e.g., improved thermostability), specific activity, protein expression, purification, processivity, strand displacement, template hopping, increased DNA / RNA affinity, and fidelity.

[0135] In one embodiment, the present disclosure relates to a non-naturally occurring enzyme that reverse transcribes a template nucleic acid molecule to produce a complementary deoxyribonucleic acid (cDNA) product, a nucleic acid product, and amplifies a cDNA product within a period of about 3 hours or less and / or with a performance index of more than about 1.0 for at least one enzyme property selected from the group consisting of: improved stability (e.g., improved thermostability), specific activity, protein expression, purification, processivity, strand displacement, template hopping, increased DNA / RNA affinity, and fidelity. In some cases, the temperature is about 25°C to about 40°C (e.g., about 28°C, about 30°C, about 32°C, about 35°C, or about 37°C). In some cases, the temperature is about 8°C to about 50°C (e.g., about 8°C, about 20°C, about 42°C, about 45°C, or about 50°C).

[0136] In one embodiment, the present disclosure relates to a non-naturally occurring enzyme that reverse transcribes a template nucleic acid molecule to produce a complementary deoxyribonucleic acid (cDNA) product and amplifies the cDNA product within a period of 3 hours or less (e.g., 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, or 30 minutes or less) and / or for a given nucleotide substrate with a processivity that is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 95%, or at least about 98% higher than the processivity of a reference enzyme for the same nucleotide substrate.

[0137] In one embodiment, the present disclosure relates to a non-naturally occurring enzyme that reverse transcribes a template nucleic acid molecule to produce a nucleic acid product and amplifies the nucleic acid product within a period of 3 hours or less (e.g., 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, or 30 minutes or less) and / or for a given nucleotide substrate with a processivity that is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 95%, or at least about 98% higher than the processivity of a reference enzyme for the same nucleotide substrate.

[0138] In one embodiment, the present disclosure provides a method for amplifying a nucleic acid molecule, comprising performing nucleic acid amplification on a nucleic acid molecule using a modified reverse transcriptase. In some cases, the reverse transcriptase is capable of amplifying a nucleic acid molecule with a processivity of at least about 80%, at least about 88%, at least about 90%, at least about 95%, or at least about 98% per base at about 4°C, about 8°C, about 12°C, about 30°C, about 28°C, about 29°C, about 32°C, about 35°C, about 37°C, about 42°C, about 50°C, or above about 42°C.

[0139] Method for preparing RNA library

[0140] In some aspects, the present disclosure provides a method for preparing complementary deoxyribonucleic acid (cDNA) molecules, comprising: partitioning a cell and a non-naturally occurring reverse transcriptase, the cell comprising ribonucleic acid (RNA) molecules; releasing the RNA molecules from the cell in the partition; and synthesizing a complementary deoxyribonucleic acid (cDNA) library from the RNA molecules using the non-naturally occurring reverse transcriptase in the partition, the non-naturally occurring transcriptase synthesizing the cDNA library with a processivity of 20 nucleotides or more per consecutive operation, whereby processivity is defined as the number of reactants produced in one consecutive operation without dissociation. In some aspects, the processivity of the enzyme is about 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides or more per second.

[0141] In some aspects, the non-naturally occurring reverse transcriptase has at least 80% identity to SEQ ID NOs: 1-20. In some aspects, the partition further comprises: one or more acceptor nucleic acid molecules; and a non-naturally occurring reverse transcriptase, wherein the non-naturally occurring reverse transcriptase has at least 80% identity to SEQ ID NOs: 1-20.

[0142] Current methods for performing single cell library preparation and low input methods include a variety of constrained methods, such as emulsion-based techniques, nanofabrication-based techniques, cell sorting techniques, and serial dilution-based techniques. Single cell library preparation and low input methods include many challenges, including but not limited to the risk of artifactual amplification due to excess amounts of reagents such as oligomeric linkers and primers rather than the RNA sample itself. Achieving quality library preparation requires high RNA to DNA conversion efficiency and low oligomeric linker-linker products.

[0143] In some aspects, the present disclosure provides a method for preparing complementary deoxyribonucleic acid (cDNA) molecules for single cell or low RNA sample input, wherein the RNA sample input is 5 to 50 pg, 10 to 50 pg, 15 to 50 pg, 20 to 50 pg, 25 to 50 pg, 30 to 50 pg, 35 to 50 pg, 40 to 50 pg, 45 to 50 pg.

[0144] In some aspects, the present disclosure provides an enzyme platform for preparing complementary deoxyribonucleic acid (cDNA) molecules for single cell or low RNA sample input, which provides the necessary high RNA sample library conversion efficiency. In some cases, the methods disclosed herein provide a relatively simple protocol with a small number of steps, ensuring a small amount of sample loss.

[0145] In some aspects, the present disclosure provides an enzymatic platform for preparing complementary deoxyribonucleic acid (cDNA) molecules for single cell or low RNA sample input, which is not limited to target polyadenylated ribonucleic acid (RNA) from cells. In some cases, the enzymatic platform disclosed herein captures non-polyadenylated RNA, such as microRNA (miRNA), non-coding RNA (ncRNA), long intergenic non-coding RNA (lincRNA), long non-coding RNA (InRNA). In some cases, the enzymatic platform disclosed herein captures the entire transcriptome, thus, including but not limited to messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), piwi interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and rDNA-derived small RNA (srRNA).

[0146] In one embodiment, the disclosure relates to a method for preparing a concatemer of nucleic acid molecules. In some cases, the method includes processing the ends of multiple double-stranded nucleic acid molecules. In some cases, the method includes adding a first plurality of joint molecules to multiple double-stranded nucleic acid molecules. In some cases, the first plurality of joint molecules include one or more overhang sequences. In some cases, at least two of the one or more overhang sequences are complementary to each other. In some cases, the method provides a double-stranded nucleic acid molecule connected by a first plurality of joints. In some cases, the method includes adding a polymerase (e.g., adding a polymerase to the double-stranded nucleic acid molecule connected by the first plurality of joints). In some cases, a polymerase is added in the absence of a primer. In some cases, the method does not include adding a primer. In some cases, a polymerase forms a double-stranded nucleic acid concatemer connected by a first group of joints. In some cases, a double-stranded nucleic acid concatemer connected by a first group of joints is formed by joining two or more double-stranded nucleic acid molecules connected by one or more overhang sequences. In some cases, the method includes adding a second plurality of joint molecules to the first group (e.g., a first joint molecule). In some cases, a second plurality of joint molecules include one or more overhang sequences. In some cases, at least two of the one or more overhang sequences are complementary to each other. In some cases, the method provides a second set of adapter-ligated double-stranded nucleic acid molecules.In some cases, any of the previous cases can be repeated using a set of adapter molecules to obtain concatemers comprising a predetermined average length.

[0147] In one embodiment, the disclosure relates to a method for preparing a concatemer of nucleic acid molecules. In some cases, the method comprises end repairing at least one nucleic acid molecule and / or multiple double-stranded nucleic acid molecules. In some cases, the method comprises adding at least one or more joint molecules to at least one nucleic acid molecule and / or multiple double-stranded nucleic acid molecules. In some cases, adding at least one or (first) multiple joint molecules to at least one nucleic acid molecule and / or multiple double-stranded nucleic acid molecules comprises joining. In some cases, adding at least one or (first) multiple joint molecules to at least one nucleic acid molecule and / or multiple double-stranded nucleic acid molecules comprises reverse transcriptase (e.g., R2 reverse transcriptase or modified reverse transcriptase). In some cases, in some cases, at least one or more joint molecules comprise one or more overhang sequences. In some cases, at least two overhang sequences are complementary to each other (e.g., double-stranded nucleic acid molecules connected by (first) multiple joints are provided). In some cases, at least one or more joint molecules comprise a sequence (e.g., overhang sequence) connected / joined to the 3' end of the nucleic acid molecule and / or a sequence (e.g., overhang sequence) connected / joined to the 5' end of the nucleic acid molecule. In some cases, the nucleic acid molecule comprises a joint on the 3' end and the 5' end. In some cases, the joint bound to the 3' end is complementary to the joint bound to the 5' end. In some cases, the joint sequence is unknown. In some cases, the joint sequence is predetermined. In some cases, the joint serves as a template and / or primer. In some cases, the joint bound to the 3' end of a nucleic acid molecule can be bound to the joint on the 5' end of another nucleic acid molecule. In some cases, the method further comprises adding a polymerase to the joint connected to the nucleic acid molecule. In some cases, the method further comprises adding a polymerase to the double-stranded nucleic acid molecules connected to (first) multiple joints. In some cases, a polymerase is added in the absence of a primer. In some cases, a polymerase is joined to the double-stranded nucleic acid molecules connected to form a first group of joints connected double-stranded nucleic acid concatemers by one or more overhang sequences of the double-stranded nucleic acid molecules connected to two or more joints. In some cases, a polymerase allows the joint connected to the nucleic acid molecule to form a concatemer. In some cases, the method comprises adding a second plurality of joint molecules to the first group. In some cases, the second plurality of joint molecules comprises one or more overhang sequences. In some cases, at least two overhang sequences are complementary to each other. In some cases, a second group of joints connected double-stranded nucleic acid molecules are formed. In some cases, the concatemer length or the number of templates connected can be determined, for example, by tagging the adapter with modified nucleotides (e.g., by introducing methylated nucleotides or by inserting dUTP). In some cases, the length of the concatemer can be adjusted based on the ratio between modified / unmodified adapters. In some cases, the adapter sequence can serve as a homology priming site (annealing to the homology point ssDNA fragment, serving as template and primer).In some cases, the method includes amplifying concatemers by PCR or isothermal reaction. In some cases, the reaction in PCR undergoes a selected number of cycles (the more cycles, the longer the concatemers) or time (isothermal amplification). In some cases, the reaction is stopped and the (long) dsDNA concatemers are joined with two unique dsDNA joints. In some cases, the length of the concatemers can be manipulated. In some cases, the length of the concatemers can be determined at least based on the number of PCR cycles and / or the amount of time (e.g., time in isothermal amplification) and / or based on the modified nucleotides present in the joint. In some cases, the joint comprises a unique molecule recognition sequence (UMI). In some cases, the polymerase joins the double-stranded nucleic acid molecules connected by two or more joints in a PCR or isothermal amplification reaction. In some cases, the joint comprises at least one modified nucleotide.

[0148] In some cases, the method for preparing a nucleic acid library and / or a complementary cDNA library comprises preparing the library in at most about 1 hour, at most about 2 hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, at most about 7 hours, at most about 10 hours, at most about 15 hours, or at most about 20 hours.

[0149] In one embodiment, the present disclosure relates to methods and processes capable of discovering novel markers and mutations for cancer, and / or providing methods for precision medicine. In some cases, the methods and processes disclosed herein provide a higher sensitivity of <0.1% for capturing minor alleles in ctDNA (the available current methods have a sensitivity of >1%). In some cases, the methods and / or processes disclosed herein comprise 1 pot (e.g., a single container), 1 step protocol, and the library is prepared from a sample in an amount of time equal to or less than about 2 hours.

[0150] The present disclosure relates to a method for preparing a complementary deoxyribonucleic acid (cDNA) molecule. In some cases, the method comprises annealing a primer to a template nucleic acid molecule, thereby producing an annealed template nucleic acid molecule. In some cases, the method further comprises mixing the annealed template nucleic acid molecule, one or more receptor nucleic acid molecules and a modified reverse transcriptase in the presence of nucleotides. In some cases, a modified reverse transcriptase produces a plurality of continuous complementary deoxyribonucleic acid molecules. In some cases, a plurality of continuous complementary deoxyribonucleic acid molecules are prepared within a maximum of about 2 hours. In some cases, a plurality of continuous complementary deoxyribonucleic acid molecules are produced by reversely transcribing the sequence of the annealed template nucleic acid molecule by a modified reverse transcriptase. In some cases, the modified reverse transcriptase then migrates to a receptor nucleic acid molecule (e.g., one or more receptor nucleic acid molecules). In some cases, a reverse transcriptase (e.g., a modified reverse transcriptase) is capable of reversely transcribing the sequence of a template and / or receptor nucleic acid molecule at a temperature of about 12°C to about 42°C. In some cases, the reverse transcriptase (e.g., a modified reverse transcriptase) is capable of reverse transcribing the sequence of the template and / or the receptor nucleic acid molecule at a temperature of about 8°C to about 50°C (e.g., about 8°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 48°C). In some cases, the reverse transcriptase (e.g., a modified reverse transcriptase) is capable of reverse transcribing the sequence of the template and / or the receptor nucleic acid molecule at a temperature of up to about 4°C, up to about 8°C, up to about 15°C, up to about 20°C, up to about 25°C, up to about 30°C, up to about 35°C, up to about 40°C, up to about 45°C, or up to about 48°C. In some cases, reverse transcription occurs at an error rate of up to about 5%. In some cases, the reverse transcriptase (e.g., modified reverse transcriptase) can reverse transcribe the template and / or receptor nucleic acid molecule with an error rate of up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, or up to about 1%. In some cases, the reverse transcriptase (e.g., modified reverse transcriptase) can migrate from the template to the receptor nucleic acid molecule, regardless of the sequence identity between the template and the receptor nucleic acid molecule. In some cases, the method is prepared in a single container. In some cases, the template nucleic acid molecule is a fragmented DNA template, a fragmented RNA template, a non-fragmented DNA template, a non-fragmented RNA template, or a combination thereof. In some cases, the method further comprises adding a tag to the template nucleic acid molecule, thereby generating a plurality of continuous complementary deoxyribonucleic acid molecules with tags. In some cases, the method further comprises performing a polymerase chain reaction amplification reaction, thereby forming one or more amplicons.

[0151] The present disclosure relates to methods for preparing complementary deoxyribonucleic acid (cDNA) molecules using modified reverse transcriptases. In some cases, the method for preparing cDNA molecules is performed by template jumping. In some cases, the modified reverse transcriptase has improved enzyme properties compared to a naturally occurring or unmodified or wild-type enzyme (e.g., a wild-type reverse transcriptase). In some cases, the method for preparing cDNA molecules comprises: (a) annealing a primer to a template; and (b) mixing the template annealed to the primer with a modified reverse transcriptase and a receptor nucleic acid molecule (e.g., a receptor RNA, DNA, or a combination thereof) in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce a cDNA molecule complementary to the template and / or a receptor nucleic acid molecule. In some cases, the enzyme (e.g., a modified reverse transcriptase) produces continuous cDNA molecules by migrating from the template to the receptor nucleic acid molecule. In some cases, template jumping is independent of the sequence identity between the template and the receptor nucleic acid molecule. In some cases, steps (a) and (b) are performed simultaneously. In some cases, step (a) comprises step (b) (e.g., step (a) and step (b) are combined into one step). In some cases, at least one of step (a) and / or step (b) further comprises adding a hot start thermostable polymerase. In some cases, the method of the present disclosure is performed in a single test tube. In some cases, the method of the present disclosure further comprises a polymerase chain reaction (PCR) amplification reaction. In some cases, the PCR amplification reaction is performed in a single test tube (e.g., the same test tube from steps (a) and (b)). In some cases, all steps of the method of the present disclosure are performed in a single test tube.

[0152] The present disclosure relates to a method for preparing a nucleic acid molecule concatemer for sequencing. In some cases, the method includes joining a nucleic acid molecule with a first joint. In some cases, the method further includes forming a concatemer by amplifying the joined nucleic acid molecule by performing a nucleic acid amplification reaction. In some cases, an amplification reaction is performed in the absence of a primer. In some cases, the method further includes joining a concatemer with a second joint. In some cases, the joint (first and / or second joint) is designed to allow molecules (such as nucleic acid molecules and / or templates and / or primers and / or receptors) to anneal and extend based on recombination or homology. In some cases, the nucleic acid amplification reaction is a polymerase chain reaction (PCR) or isothermal amplification. In some cases, the first joint includes a unique molecule identification (UMI) sequence. In some cases, the first joint acts as a primer. In some cases, the first joint includes a single-stranded nucleic acid. In some cases, the single-stranded nucleic acid includes single-stranded DNA (ssDNA). In some cases, the second joint includes a double-stranded nucleic acid. In some cases, the double-stranded nucleic acid includes double-stranded DNA (dsDNA). In some cases, the first joint is different from the second joint. In some cases, the first joint comprises two or more joints. In some cases, the second joint comprises two or more joints. In some cases, both ends of the nucleic acid molecule comprise joints. In some cases, only one end of the nucleic acid molecule comprises a joint. In some cases, both 3' and 5' ends of the nucleic acid molecule comprise joints.

[0153] The present disclosure relates to methods for preparing complementary deoxyribonucleic acid (cDNA) molecules using modified reverse transcriptases. In some cases, the method for preparing cDNA molecules is performed by template jumping. In some cases, the modified reverse transcriptase has improved enzyme properties compared to naturally occurring or unmodified or wild-type enzymes (e.g., wild-type reverse transcriptases). In some cases, the method for preparing cDNA molecules includes mixing primers, templates, modified reverse transcriptases, and receptor nucleic acid molecules (e.g., receptor RNA, DNA, or a combination thereof) under conditions sufficient to produce cDNA molecules complementary to templates and / or receptor nucleic acid molecules in the presence of nucleotides (e.g., dNTPs). In some cases, the method includes adding a hot start thermostable polymerase (e.g., to a mixing step). In some cases, the method of the present disclosure is performed in a single test tube. In some cases, the method of the present disclosure further includes a polymerase chain reaction (PCR) amplification reaction. In some cases, the PCR amplification reaction is performed in a single test tube (e.g., the same test tube as the mixing step). In some cases, all steps of the method of the present disclosure are performed in a single test tube (single container).

[0154] In some cases, the method for preparing a cDNA molecule comprises: (a) annealing one or more primers to a template; and (b) in the presence of nucleotides (e.g., dNTPs), mixing the template annealed to one or more primers with a modified reverse transcriptase and a receptor nucleic acid molecule (e.g., receptor RNA, DNA, or a combination thereof) under conditions sufficient to produce a cDNA molecule complementary to the template and / or receptor nucleic acid molecule. In some cases, the method for preparing a cDNA molecule is performed by template hopping. In some cases, steps (a) and (b) are performed simultaneously. In some cases, step (a) comprises step (b) (e.g., step (a) and step (b) are combined into one step). In some cases, at least one of steps (a) and / or step (b) further comprises adding a hot start thermostable polymerase. In some cases, the method of the present disclosure is performed in a single test tube. In some cases, the method of the present disclosure further comprises a polymerase chain reaction (PCR) amplification reaction. In some cases, the PCR amplification reaction is carried out in a single tube (e.g., the same tube used in or from steps (a) and (b)). In some cases, all steps of the disclosed method are performed in a single tube (one pot or single pot).

[0155] In some cases, the method for preparing cDNA molecules is included in the presence of nucleotides (e.g., dNTPs), under conditions sufficient to produce cDNA molecules complementary to template and / or receptor nucleic acid molecules, one or more primers, templates, modified reverse transcriptases and receptor nucleic acid molecules (e.g., receptor RNA, DNA, or a combination thereof) are mixed. In some cases, the method for preparing cDNA molecules is performed by template hopping. In some cases, the method includes adding a hot start thermostable polymerase (e.g., to a mixing step). In some cases, the method of the present disclosure is performed in a single test tube. In some cases, the method of the present disclosure further includes polymerase chain reaction (PCR) amplification reaction. In some cases, the PCR amplification reaction is performed in a single test tube (e.g., the same test tube as the mixing step). In some cases, all steps of the method of the present disclosure are performed in a single test tube.

[0156] ssDNA sponge for RNA depletion

[0157] Ribosomal RNA can constitute up to 80% or more of the total RNA in a sample. It is usually necessary to separate mRNA from rRNA because rRNA may adversely affect the quantitative analysis of mRNA. A method for separating rRNA from other RNA biotypes, including mRNA, miRNA, lncRNA and lincRNA, is to deplete rRNA from a sample. An example is to hybridize rRNA molecules using oligonucleotides, such as oligonucleotides homologous to 5.8SrRNA, 17S rRNA, 18S rRNA or 28S rRNA in the case of eukaryotic rRNA or to 5S rRNA, 16S rRNA or 23S rRNA in the case of bacterial rRNA. Oligonucleotides are designed so that they can be "captured" and hybridization products can be removed from the sample. For example, oligonucleotides can be fixed on the surface of, for example, a column or a bead. MICROB Express (registered trademark) and MICROBEnrich (registered trademark) (Ambion, Austin, Tex.) are examples of commercially available kits for depleting rRNA. Methods and compositions for depleting rRNA from a sample are described in U.S. Application No. 10 / 029,397, which is incorporated by reference. The poly(A) tail at the 3' end of most eukaryotic mRNAs can be used to separate these molecules from rRNA and other non-mRNA species that lack such a poly(A) tail.

[0158] Rather than removing rRNA from a sample, in some cases, the methods of the present disclosure encompass preventing RNA from undergoing any possible amplification or additional reactions. In some cases, the present disclosure provides a method for processing a sample containing messenger RNA (mRNA), ribosomal RNA (rRNA) molecules, microRNA (miRNA), long non-coding RNA (IncRNA), long intergenic non-coding RNA (lincRNA) and other RNA biotypes, comprising synthesizing complementary deoxyribonucleic acid (cDNA) molecules using such mRNA molecules or other RNA biotypes, including miRNA, IncRNA and lincRNA, in the presence of such rRNA molecules that are prevented from transcription, such that less than 30% of such cDNA molecules contain sequences from such rRNA molecules.

[0159] In some aspects, the present disclosure provides a method for processing a sample comprising messenger ribonucleic acid (mRNA) and ribosomal ribonucleic acid (rRNA) molecules, comprising synthesizing complementary deoxyribonucleic acid (cDNA) molecules using the mRNA molecules in the presence of the rRNA molecules that are blocked from transcription, such that less than 30% of the cDNA molecules contain sequences from the rRNA molecules.

[0160] In some aspects, the present disclosure provides a method for processing a mixture comprising messenger ribonucleic acid (mRNA) and ribosomal ribonucleic acid (rRNA) molecules, comprising: in the mixture, splitting the ribosomal ribonucleic acid (rRNA) molecules to obtain a plurality of rRNA fragments; contacting one or more single-stranded nucleic acid sequences with the plurality of rRNA fragments, the one or more single-stranded nucleic acid sequences being complementary to at least a subset of the rRNA fragments, thereby providing one or more rRNA fragment complexes comprising the one or more single-stranded nucleic acid sequences hybridized to at least the subset of the rRNA fragments; and synthesizing at least one complementary deoxyribonucleic acid (cDNA) molecule from the mRNA using a reverse transcriptase in the presence of the one or more rRNA fragment complexes (Figures 5 and 6).

[0161] In some aspects, the ssDNA complementary to the rRNA (DNA-sponge) has a linear form with a blocked 3' end. In some cases, the DNA-sponge has a circular form. In some cases, the DNA-sponge is concatemerized. FIG. 5 shows a DNA-sponge with a circular form, while Figure 5B Rolling circle amplification (RCA) products are shown. This figure shows the function of DNA-sponge, which is annealed to rRNA fragments. Annealing of rRNA fragments to large complementary ssDNA makes the 3' end of the rRNA fragment unavailable for poly A polymerase or for 3'-priming by R2 enzyme. Thus, rRNA fragments without available 3'-ends will not be converted into sequencing libraries, as shown in Figure 5.

[0162] Direct RNA and ssDNA sequencing using R2 enzyme

[0163] In some aspects, the present disclosure provides a method for sequencing a single-stranded nucleic acid molecule, comprising: providing a reaction mixture comprising the single-stranded nucleic acid molecule and a non-naturally occurring enzyme, wherein the non-naturally occurring enzyme comprises: a finger domain derived from an R2 retrotransposon, a palm domain derived from an R2 retrotransposon, a thumb domain derived from an R2 retrotransposon, and an endonuclease domain derived from an R2 retrotransposon. In some cases, the method disclosed herein comprises subjecting the reaction mixture to conditions sufficient to use the non-naturally occurring enzyme to incorporate individual nucleotides into a growing chain complementary to the single-stranded nucleic acid molecule, wherein the individual nucleotides are incorporated into the growing chain to obtain a detectable signal. In some cases, the method disclosed herein comprises detecting the detectable signals, thereby sequencing the single-stranded nucleic acid molecule.

[0164] In some aspects, the present disclosure provides a method for sequencing a single-stranded nucleic acid molecule, wherein the single-stranded nucleic acid molecule is an RNA molecule. In some cases, the methods disclosed herein are used to sequence a single-stranded nucleic acid molecule, wherein the single-stranded nucleic acid molecule is a single-stranded DNA molecule.

[0165] In some aspects, the present disclosure provides a method for sequencing a single-stranded nucleic acid molecule, wherein the conditions sufficient to directly sequence the single-stranded nucleic acid molecule comprise optics-based single molecule sequencing conditions.

[0166] In some aspects, the present disclosure provides a method for sequencing a single-stranded nucleic acid molecule, wherein the conditions sufficient to directly sequence the single-stranded nucleic acid molecule comprise microscopy-based single molecule sequencing conditions.

[0167] In some aspects, the present disclosure provides a method for sequencing a single-stranded nucleic acid molecule, wherein the conditions sufficient to directly sequence the single-stranded nucleic acid molecule comprise nanopore-based single molecule sequencing conditions.

[0168] In some aspects, the present disclosure provides a method for sequencing a single-stranded nucleic acid molecule, wherein the conditions sufficient to directly sequence the single-stranded nucleic acid molecule comprise field effect transistor-based single molecule sequencing conditions.

[0169] Commonly used single molecule sequencing techniques are based on long reads, where several kb fragments are read as one continuous read. In some aspects, the present disclosure provides a method in which the enzymes disclosed herein are capable of efficient template hopping, thereby allowing sequencing of a large number of templates in a single continuous sequencing operation.

[0170] In situ RNA sequencing using non-naturally occurring enzymes

[0171] Unlike in situ RNA sequencing, conventional RNA sequencing provides a summary analysis of gene expression across the entire transcriptome, but still lacks spatial context. However, in situ RNA sequencing allows genome-wide profiling of gene expression in situ in fixed cells and fixed tissues ( Figure 6 ).

[0172] In some aspects, the present disclosure provides a method comprising preparing complementary deoxyribonucleic acid (cDNA) molecules from one or more ribonucleic acids (RNA) using a non-naturally occurring enzyme, wherein the one or more ribonucleic acids (RNA) are derived from an in situ tissue of a subject or from a fixed ex vivo tissue of the subject, wherein the non-naturally occurring enzyme comprises a palm and finger domain derived from an R2 retrotransposon, a palm domain derived from an R2 retrotransposon, a thumb domain derived from an R2 retrotransposon, and an endonuclease domain derived from an R2 retrotransposon; thereby generating cDNA molecules from the in situ tissue of the subject or from the fixed ex vivo tissue of the subject; and sequencing the cDNA molecules generated using the non-naturally occurring enzyme disclosed herein.

[0173] In some aspects, the present disclosure provides a method comprising preparing complementary deoxyribonucleic acid (cDNA) molecules from one or more ribonucleic acids (RNA) using a non-naturally occurring enzyme, wherein the one or more ribonucleic acids (RNA) are derived from an in situ tissue of a subject or from a fixed ex vivo tissue of the subject, wherein the fixed ex vivo tissue of the subject is fixed in formaldehyde.

[0174] In some aspects, the present disclosure provides a method comprising preparing complementary deoxyribonucleic acid (cDNA) molecules from one or more ribonucleic acids (RNA) using a non-naturally occurring enzyme, wherein the one or more ribonucleic acids (RNA) are derived from an in situ tissue of a subject or from a fixed ex vivo tissue of the subject, wherein the fixed ex vivo tissue of the subject is fixed and embedded in paraffin.

[0175] In some aspects, the methods disclosed herein consist of labeling cDNA with a barcode including but not limited to spatial information ( Figure 6 ) Then the cDNA can be converted into a sequencing library. In some cases, the spatial specific barcoding technology consists of a glass plate with oligonucleotide primers printed in a spatial specific manner. In some cases, the primer used in the method disclosed herein is a poly-T oligonucleotide of specific barcoding. In some aspects, the method disclosed herein is highly sensitive and can be operated under extremely low sample input. In some aspects, the method disclosed herein has a scheme using random primers. In some cases, the method disclosed herein has a scheme using specific primers.

[0176] In some cases, the biological sample has been purified. In some cases, the biological sample has not been purified. In some cases, when the biological sample is provided to the test tube, the nucleic acid of the biological sample has not been extracted. For example, when the biological sample is provided to the test tube, the RNA or DNA in the biological sample may not have been extracted from the biological sample. In some cases, before the biological sample is provided to the reaction vessel (e.g., test tube), the target nucleic acid (e.g., target RNA or target DNA) present in the biological sample may not be concentrated. Any suitable biological sample containing nucleic acid can be obtained from the subject.

[0177] In some cases, amplify nucleic acids from biological samples obtained from subjects. In some cases, biological samples are obtained directly from subjects. In some cases, biological samples obtained directly from subjects refer to biological samples that have been further processed after being obtained from subjects. In some cases, biological samples obtained directly from subjects refer to biological samples that have not been further processed after being obtained from subjects, but any method can be used to collect biological samples from subjects for further processing. For example, blood is obtained directly from subjects by accessing the circulatory system of the subject, taking blood from the subject (e.g., via a needle) and injecting the taken blood into a container. The container may contain reagents (e.g., anticoagulants) so that the blood sample is suitable for further analysis. In another example, a swab can be used to obtain epithelial cells on the surface of the oropharynx of a subject. After obtaining a biological sample from a subject, a swab containing a biological sample can be contacted with a fluid (e.g., a buffer) to collect biological fluids from the swab.

[0178] The present disclosure relates to a method for detecting, diagnosing and / or prognosing a disease (e.g., cancer) in a subject, comprising: (a) obtaining sequence information of a nucleic acid sample (e.g., a free nucleic acid sample) derived from a subject, and (b) detecting circulating tumor nucleic acids in the sample using the sequence information derived from step (a). In some cases, obtaining sequence information according to step (a) comprises using one or more joints. In some cases, one or more joints comprise a molecular barcode. The joint may comprise one or more terminal modifications. The joint may comprise a 5' phosphate. The joint may comprise two 5' phosphates. The joint may comprise a 3' hydroxyl. The joint may comprise two 3' hydroxyls. The joint may lack a 3' hydroxyl.

[0179] In some cases, the molecular barcode comprises a random sequence. In some cases, the method is capable of detecting less than or equal to about 0.75%, 0.50%, 0.25%, 0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0005%, or 0.0 0001%, 1%, 1.75%, 1.5%, 1.25%, 2%, 3%, 4%, 5%, 6%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% cell-free nucleic acid. In some cases, the method is capable of detecting less than or equal to about 0.75%, 0.50%, 0.25%, 0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0005%, or 0.00001%, 1%, 1.75%, 1.5%, 1.25%, 2%, 3%, 4%, 5%, 6%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of circulating tumor nucleic acids. In some cases, the method is capable of detecting circulating tumor nucleic acid (ct nucleic acid) at a percentage of less than or equal to 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.50%, 0.25%, 0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0005% or 0.00001% of total free nucleic acid.In some cases, the sequence information comprises information about at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 70, 80, 100, 200, or 300 genomic regions. In some cases, the sequence information comprises information about partial all, nearly all, or all of the genome sequencing. In some cases, a concentration as low as 50 ng cfDNA can provide complete genome sequencing.

[0180] In some cases, the method of the present disclosure can be used to determine the presence of a disease (e.g., cancer) in a subject. In some cases, determining the presence of a cancer in a subject comprises obtaining a sample from a subject according to any method described herein and detecting nucleic acid molecules (e.g., nucleic acid fragments) in the sample. In some cases, determining the presence of a disease (e.g., cancer) in a subject comprises amplifying and / or sequencing nucleic acid molecules. In some cases, the presence of nucleic acid molecules indicates cancer. In some cases, the presence of nucleic acid molecules indicates prenatal status. In some cases, nucleic acid molecules and / or templates include unknown sequences. In some cases, the sample is a biological sample. In some cases, the biological sample includes circulating tumor DNA.

[0181] In some cases, the biological sample comprises a tissue sample.

[0182] In some cases, the methods of the present disclosure comprise detecting an amplicon produced by an amplification primer, wherein the presence of the amplicon determines the presence of a modified reverse transcriptase in the sample.

[0183] In some cases, the methods of the present disclosure comprise providing a prenatal diagnosis based on the presence or absence of a nucleic acid molecule (eg, a cDNA molecule).

[0184] Preparation of RNA libraries for sequencing

[0185] In some aspects, the present disclosure provides a method for preparing complementary deoxyribonucleic acid (cDNA) molecules, comprising: fragmenting a ribonucleic acid (RNA) molecule to obtain multiple RNA fragments; removing 3'-phosphate, 2'-phosphate and cyclic 2'3'-phosphate groups from one or more of the RNA fragments, thereby producing one or more dephosphorylated fragmented rRNAs; adding a poly-A tail to the one or more dephosphorylated fragmented RNAs; adding the following to the one or more dephosphorylated fragmented RNAs: a primer adapter comprising an oligo-T sequence, a poly-T and another adapter sequence compatible with a major sequencing technology; an acceptor adapter; and a non-naturally occurring R2 enzyme having a processivity of 20 nucleotides or longer nucleotides.

[0186] In some aspects, the present disclosure provides a method for preparing a complementary deoxyribonucleic acid (cDNA) molecule, comprising a non-naturally occurring R2 enzyme, wherein the non-naturally occurring R2 enzyme reverse transcribes a sequence from the one or more dephosphorylated fragmented RNAs in a 3' to 5' order, wherein upon reaching the 5' end of the one or more dephosphorylated fragmented RNAs the R2 enzyme jumps to the 3'-end of the acceptor adaptor.

[0187] In some aspects, the methods disclosed herein consist of an acceptor linker comprising a nucleotide analog ( Fig. 9 ). In some cases, the nucleotide analog is at the 5' end of the acceptor linker. In some cases, the acceptor linker comprises a 3'-dideoxynucleotide at the 3'-end of the acceptor linker.

[0188] In some aspects, the methods disclosed herein comprise a non-naturally occurring R2 enzyme, wherein the non-naturally occurring R2 enzyme reverse transcribes a sequence from the one or more fragmented RNAs in a 3' to 5' order, wherein upon reaching the 5' end of the one or more fragmented RNAs the R2 enzyme jumps to the 3'-end of the acceptor adaptor ( Fig.10 , 11 and 12).

[0189] In some cases, primers can include adapter sequences. In some cases, the 5' tail sequence of primers includes a sequence that does not hybridize with the target (adapter sequence). The adapter sequence can be selected so that it is identical in a variety of primers with different 3' target binding sequences (i.e., "universal" 5' tail sequence). The adapter sequence is compatible with the main sequencing technologies including but not limited to Illumina, Ion Torrent, PacBio and Roche 454. This allows a single reporter probe sequence to be used to detect any desired target sequence, which is an advantage because the synthesis of the reporter probe is more complicated due to labeling. In some cases, primers can include RNA primers. In some cases, primers can include DNA primers. In some cases, primers can include R2 RNA primers. In some cases, primers can include one or more random primers.

[0190] The present disclosure relates to a method for preparing a nucleic acid molecule, comprising: mixing a fragment or degraded template (e.g., a nucleic acid fragment), a primer, a modified reverse transcriptase, and a receptor nucleic acid molecule in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce a nucleic acid molecule. In some cases, the receptor nucleic acid molecule comprises a modified nucleotide. In some cases, primer extension stops at a modified nucleotide. In some cases, the modified reverse transcriptase comprises at least one improved enzymatic property relative to a wild-type, naturally occurring, or unmodified reverse transcriptase. In some cases, the primer is an RNA primer. In some cases, the primer is an engineered primer (e.g., an engineered RNA primer). In some cases, the primer is optimized. In some cases, the primer is an optimized and / or engineered primer (e.g., an optimized and / or engineered RNA primer). In some cases, the primer is an RNA R2 primer. In some cases, the method for preparing a nucleic acid molecule is performed by template jumping. In some cases, the mixing step of the method of the present disclosure further comprises adding a hot start thermostable polymerase. In some cases, the method of the present disclosure is performed in a single test tube. In some cases, the method of the present disclosure further comprises a polymerase chain reaction (PCR) amplification reaction. In some cases, the PCR amplification reaction is carried out in the same single test tube. In some cases, all steps of the method of the present disclosure are performed in a single test tube.

[0191] The present disclosure relates to a method for preparing a nucleic acid molecule, comprising: mixing a fragment or degraded template (e.g., a nucleic acid fragment), a donor complex, a modified reverse transcriptase, and an acceptor nucleic acid molecule in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce nucleic acid molecules. In some cases, the acceptor nucleic acid molecule comprises a modified nucleotide. In some cases, primer extension stops at a modified nucleotide. In some cases, the modified reverse transcriptase comprises at least one improved enzymatic property relative to a wild-type or naturally occurring or unmodified reverse transcriptase. In some cases, the donor complex comprises a template and a primer. In some cases, the donor complex is a donor R2 complex. In some cases, the donor R2 complex comprises an RNA R2 primer. In some cases, the method for preparing a nucleic acid molecule is performed by template jumping. In some cases, the mixing step of the method of the present disclosure further comprises adding a hot start thermostable polymerase. In some cases, the method of the present disclosure is performed in a single test tube. In some cases, the method of the present disclosure further comprises a polymerase chain reaction (PCR) amplification reaction. In some cases, the PCR amplification reaction is performed in the same single tube (e.g., the same single tube used to prepare the nucleic acid molecule). In some cases, all steps of the methods of the present disclosure are performed in a single tube.

[0192] The present disclosure relates to methods for preparing complementary deoxyribonucleic acid (cDNA) libraries using modified reverse transcriptases. In some cases, the methods for preparing cDNA libraries use template jumping. In some cases, the modified reverse transcriptase has improved enzyme properties compared to a naturally occurring or wild-type or unmodified enzyme (e.g., a wild-type reverse transcriptase). In some cases, the methods for preparing cDNA libraries include: (a) annealing a primer or one or more primers to a template; and (b) mixing the template annealed to the primer or the template annealed to the one or more primers with a modified reverse transcriptase and a receptor nucleic acid molecule (e.g., a receptor RNA, DNA, or a combination thereof) in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce a cDNA molecule complementary to the template and / or a receptor nucleic acid molecule. In some cases, the methods for preparing cDNA libraries include mixing a primer or one or more primers, a template, a modified reverse transcriptase, and a receptor nucleic acid molecule (e.g., a receptor RNA, DNA, or a combination thereof) in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce a cDNA molecule complementary to the template and / or a receptor nucleic acid molecule. In some cases, enzyme (such as modified reverse transcriptase) produces continuous cDNA molecules by migrating from template to receptor nucleic acid molecule. In some cases, template jumping is unrelated to the sequence identity between template and receptor nucleic acid molecule. In some cases, the method further comprises amplification of cDNA molecules to produce cDNA library. In some cases, step (a) and step (b) are carried out simultaneously. In some cases, step (a) comprises step (b) (such as step (a) and step (b) are merged into one step). In some cases, at least one of the mixing step or step (a) and / or step (b) further comprises adding hot start thermostable polymerase. In some cases, the method of the present disclosure is performed in a single test tube. In some cases, the method of the present disclosure further comprises polymerase chain reaction (PCR) amplification reaction. In some cases, PCR amplification reaction is carried out in a single test tube (such as for mixing step or from mixing step, or step (a) and (b) in or from the same test tube of step (a) and (b)). In some cases, all steps of the method of the present disclosure are performed in a single test tube.

[0193] The present disclosure relates to a method for preparing a cDNA and / or DNA library, comprising mixing a fragment or degraded template (e.g., a nucleic acid fragment), a primer, a modified reverse transcriptase, and a receptor nucleic acid molecule in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce nucleic acid (e.g., cDNA and / or DNA) molecules. In some cases, the receptor nucleic acid molecule comprises a modified nucleotide. In some cases, primer extension stops at a modified nucleotide. In some cases, the modified reverse transcriptase comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some cases, the primer is an RNA R2 primer. In some cases, the method further comprises amplifying nucleic acid (e.g., cDNA and / or DNA) molecules to produce a cDNA library. In some cases, the method for preparing cDNA and / or DNA and / or nucleic acid molecules is performed by template jumping.

[0194] The present disclosure relates to a method for preparing a cDNA and / or DNA library, which comprises mixing a fragment or degraded template (e.g., a nucleic acid fragment), a donor complex, a modified reverse transcriptase, and an acceptor nucleic acid molecule under conditions sufficient to produce a nucleic acid (e.g., cDNA and / or DNA) molecule in the presence of nucleotides (e.g., dNTPs). In some cases, the acceptor nucleic acid molecule comprises a modified nucleotide. In some cases, primer extension stops at a modified nucleotide. In some cases, the modified reverse transcriptase comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some cases, the donor complex comprises a template and a primer. In some cases, the donor complex is a donor R2 complex. In some cases, the donor R2 complex comprises an RNA R2 primer. In some cases, the method further comprises amplifying a nucleic acid (e.g., cDNA and / or DNA) molecule to produce a cDNA and / or DNA library. In some cases, the method for preparing a cDNA and / or DNA and / or nucleic acid molecule molecule is performed by template jumping.

[0195] In some cases, the methods of the present disclosure may include a donor complex. In some cases, the donor complex includes a template and a primer. In some cases, the methods of the present disclosure may include a template. In some cases, the template is a fragmented and / or degraded template. In some cases, the template is not fragmented. In some cases, the template is RNA, DNA, or a combination of DNA and RNA. In some cases, the RNA is mRNA. In some cases, the template is mRNA.

[0196] The present disclosure relates to a method for preparing a library for sequencing, comprising: (a) obtaining a sample with free nucleic acid from a subject; and (b) adding a modified reverse transcriptase, a template (e.g., a nucleic acid template), nucleotides, a receptor nucleic acid molecule, and one or more primers to the nucleic acid. In some cases, the method further comprises performing an amplification reaction on the free nucleic acid (cf nucleic acid) derived from the sample to produce a plurality of amplicons. In some cases, the amplification reaction comprises 35 or less amplification cycles. In some cases, the method comprises generating a library for sequencing. In some cases, the library comprises a plurality of amplicons. In some cases, the modified reverse transcriptase is capable of template hopping and / or comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some cases, the nucleic acid is a combination of DNA, RNA, or RNA and DNA.

[0197] The present disclosure relates to a method for preparing complementary deoxyribonucleic acid (cDNA) molecules using template hopping, which comprises mixing a primer or one or more primers, a messenger RNA (mRNA) template, nucleotides, a modified reverse transcriptase, a receptor nucleic acid molecule, and a catalytic metal in a single test tube under conditions sufficient to produce continuous cDNA molecules. In some cases, the continuous cDNA molecules are complementary to the mRNA template and / or the receptor nucleic acid molecule. In some cases, the modified reverse transcriptase comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some cases, continuous cDNA molecules are produced. In some cases, the modified reverse transcriptase undergoes migration from the template to the receptor nucleic acid molecule.

[0198] The present disclosure relates to a method for preparing a library for sequencing, comprising mixing free nucleic acid, a modified reverse transcriptase, a template, nucleotides, an acceptor nucleic acid molecule, a catalytic metal, and one or more primers in a single test tube under conditions sufficient to generate a library. In some cases, the modified reverse transcriptase comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase.

[0199] In some cases, the nucleic acid molecule comprises an unknown nucleic acid sequence. In some cases, the template comprises an unknown nucleic acid sequence. In some cases, the migration of the template to the receptor nucleic acid molecule is independent of the sequence identity between the template and the receptor nucleic acid molecule. In some cases, the receptor nucleic acid molecule comprises modified nucleotides that can stop primer extension. In some cases, the free nucleic acid is free DNA (cfDNA), circulating tumor DNA (ctDNA) and / or formalin-fixed paraffin-embedded DNA (FFPE DNA) or a combination thereof.

[0200] In some cases, the disclosed method can add a hot start thermostable polymerase at or before any step of the method and / or while performing a mixing step. For example, a hot start thermostable polymerase can be added while a modified reverse transcriptase is added to the reaction. A hot start thermostable polymerase can be added while a receptor nucleic acid molecule is added, and / or while a template and / or primer and / or reverse transcriptase and / or nucleotides are added to a reaction tube. In some cases, a hot start thermostable polymerase is added before the PCR reaction begins. In some cases, a hot start thermostable polymerase is added before or while the RT reaction. In some cases, the hot start thermostable polymerase is a hot start taq polymerase. Amplification of the target nucleic acid can occur on beads. In some cases, amplification does not occur on beads. Amplification can be performed by isothermal amplification, such as isothermal linear amplification. In some cases, hot start PCR can be performed, wherein the reaction is heated to 95°C before adding the polymerase, for example, for two minutes, or the polymerase can be kept inactive until the first heating step in the 1st cycle. Hot start PCR can be used to minimize nonspecific amplification.

[0201] In some cases, PCR amplification is performed at a temperature sufficient to inactivate the reverse transcriptase. In some cases, PCR amplification is performed at a temperature sufficient to activate a hot start thermostable polymerase.

[0202] The present disclosure relates to a method for amplifying free nucleic acid molecules from a sample. In some cases, the sample is a biological sample. In some cases, the free nucleic acid molecules are subjected to a nucleic acid amplification comprising a reverse transcriptase (e.g., a modified reverse transcriptase). In some cases, the free nucleic acid molecules are subjected to a nucleic acid amplification comprising a reverse transcriptase (e.g., a modified reverse transcriptase) under conditions of specifying a continuous synthesis capacity amplification nucleic acid molecule. In some cases, the processivity is at least about 80% per base, at least about 81% per base, at least about 82% per base, at least about 83% per base, at least about 84% per base, at least about 85% per base, at least about 86% per base, at least about 87% per base, at least about 88% per base, at least about 89% per base, at least about 90% per base, at least about 91% per base, at least about 92% per base, at least about 93% per base, at least about 94% per base, at least about 95% per base, at least about 96% per base, at least about 97% per base, at least about 98% per base, at least about 99% per base, or at least about 100% per base. In some cases, processivity is performed at a temperature of about or up to about or at least about 12°C, about or up to about or at least about 13°C, about or up to about or at least about 14°C, about or up to about or at least about 15°C, about or up to about or at least about 16°C, about or up to about or at least about 17°C, about or up to about or at least about 18°C, about or up to about or at least about 19°C, about or up to about or at least about 20°C, about or up to about or at least about 21°C, about or up to about or at least about 22°C, about or up to about or at least about 23°C, about or up to about or at least about 24°C, about or up to about or at least about 25°C, about or up to about or at least about 26°C, about or up to about or at least about 27°C, about or up to about or at least about about 28°C, about or at most about or at least about 29°C, about or at most about or at least about 30°C, about or at most about or at least about 31°C, about or at most about or at least about 32°C, about or at most about or at least about 33°C, about or at most about or at least about 34°C, about or at most about or at least about 35°C, about or at most about or at least about 36°C, about or at most about or at least about 37°C, about or at most about or at least about 38°C, about or at most about or at least about 39°C, about or at most about or at least about 40°C, about or at most about or at least about 45°C, about or at most about or at least about 50°C, about or at most about or at least about 60°C, about or at most about or at least about 70°C, about or at most about or at least about 80°C, about or at most about or at least about 8°C.In some cases, the processivity is at least about 80% per base, at least about 81% per base, at least about 82% per base, at least about 83% per base, at least about 84% per base, at least about 85% per base, at least about 86% per base, at least about 87% per base, at least about 88% per base, at least about 89% per base, at least about 90% per base, at least about 91% per base, at least about 92% per base, at least about 93% per base, at least about 94% per base, at least about 95% per base, at least about 96% per base, at least about 97% per base, at least about 98% per base, at least about 99% per base, or at least about 100% per base at a temperature of about or at most about or at least about 30°C, or about or at most about or at least about 12°C, about or at most about or at least about 45°C, about or at most about or at least about 35°C. In some cases, the reverse transcriptase is a non-LTR retrotransposon or a modified non-LTR retrotransposon. In some cases, the reverse transcriptase is an R2 reverse transcriptase or a modified R2 reverse transcriptase. In some cases, the reverse transcriptase is an R2 non-LTR retrotransposon or a modified R2 non-LTR retrotransposon.

[0203] The present disclosure relates to a method for preparing a complementary deoxyribonucleic acid (cDNA) library and / or a DNA library from a plurality of single cells. In some cases, the method comprises the following steps: stripping nucleic acid from each single cell to provide a plurality of individual nucleic acid samples. In some cases, the nucleic acid in each individual nucleic acid sample is from a single cell. In some cases, the method further comprises annealing the nucleic acid template to one or more primers. In some cases, the method further comprises mixing the nucleic acid template annealed to one or more primers with an acceptor template (or acceptor nucleic acid molecule) and a modified reverse transcriptase under conditions that effectively produce cDNA and / or DNA molecules in the presence of nucleotides. In some cases, the modified reverse transcriptase is capable of template hopping and / or comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some cases, the method further comprises amplifying cDNA molecules and / or DNA molecules to produce cDNA and / or DNA libraries.

[0204] The present disclosure relates to methods for detecting nucleic acid molecules. In some cases, the method comprises mixing a sample comprising nucleic acid molecules with an acceptor template (or acceptor nucleic acid molecule), a modified reverse transcriptase, a primer and nucleotides under conditions that effectively produce nucleic acid molecules. In some cases, the modified reverse transcriptase comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some cases, the acceptor template (or acceptor nucleic acid molecule) comprises at least one modified nucleotide. In some cases, the modified nucleotide can stop primer extension. In some cases, the method further comprises amplifying nucleic acid molecules.

[0205] The present disclosure relates to any method disclosed herein, wherein the method may further comprise detecting at least one amplicon produced by the amplification primer. In some cases, the presence of at least one amplicon indicates the presence of at least one modified reverse transcriptase in the sample.

[0206] In some cases, any method of the present disclosure does not include a purification step. In some cases, any method of the present disclosure includes at least one purification step. In some cases, any method of the present disclosure includes at least two purification steps. In some cases, any method of the present disclosure includes at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, or at least twenty purification steps.

[0207] The present disclosure relates to a method for preparing a library for sequencing.

[0208] In some cases, the modified reverse transcriptase is a modified non-retroviral reverse transcriptase. In some cases, the modified reverse transcriptase is a modified non-LTR retrotransposon. In some cases, the modified reverse transcriptase is a modified R2 reverse transcriptase.

[0209] In some cases, the sample is a biological sample. In some cases, the biological sample comprises circulating tumor DNA. In some cases, the biological sample comprises a tissue sample. In some cases, the nucleic acid is from the sample. In some cases, the sample is a liquid biopsy sample. In some cases, the sample can be an RNA sample. In some cases, the RNA sample can be used for various purposes, including but not limited to PCR, conjugation, transcriptome analysis, microarray analysis, Northern blot analysis and cDNA library construction. In some cases, the present disclosure is directed to a method for amplifying a cDNA library from a relatively low amount of cells and / or single cells in an amount and quality suitable for transcriptome analysis or microarray analysis by, for example, sequencing.

[0210] In some cases, nucleic acid and / or template have unknown sequence. In some cases, nucleic acid and / or template are RNA, DNA or a combination of RNA and DNA. In some cases, RNA is mRNA. In some cases, mRNA comprises internal initiation. In some cases, nucleic acid can be fragmented nucleic acid and / or degraded nucleic acid. In some cases, template can be fragmented template and / or degraded template. In some cases, nucleic acid can be non-fragmented nucleic acid and / or non-degraded nucleic acid. In some cases, template can be non-fragmented template and / or non-degraded template. In some cases, nucleic acid and / or template indicate disease. In some cases, nucleic acid and / or template indicate cancer. In some cases, nucleic acid is equal to or less than about 0.01 micromolar. In some cases, nucleic acid is between about 0.1nM to about 100nM and. In some cases, nucleic acid is equal to or less than about 500 femtomolar.

[0211] In some cases, RNA is obtained from a source selected from the group consisting of single cells, cultured cells, tissues, RNA transcription-based amplified RNA (e.g., TTR amplified RNA or other DNA-dependent RNA polymerase transcribed RNA), RNA-promoter-driven transcribed RNA, aRNA, aRNA amplified RNA, single cell mRNA library, isolated mRNA, RNA contained in cells, and a combination of RNA sources. In some cases, RNA is prepared from a plurality of fixed cells, wherein the fixed cells are protected from RNA degradation and undergo permeabilization for enzyme penetration. In some cases, fixed cells are obtained from fixative-treated cultured cells, frozen fresh tissue, fixative-treated fresh tissue, or paraffin-embedded tissue on a slide.

[0212] In some cases, RNA molecules can be products of in vitro synthesis, or can be isolated from cells or tissues (Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, Wiley, 1995). Cells and tissues suitable for obtaining RNA useful in practicing the present disclosure can include animal cells and plant cells. In some cases, cells include mammalian cells and insect cells. RNA can also be isolated from prokaryotic cells, such as bacteria.

[0213] In some cases, the template is RNA, DNA or a combination of RNA and DNA. In some cases, the template can be a fragmented template and / or a degraded template. In some cases, the template is not degraded and / or fragmented. In some cases, RNA is mRNA. In some cases, the template is an RNA template. In some cases, the template is a DNA template. In some cases, the template is a DNA and / or RNA template. In some cases, the template is a mixture of DNA and RNA. In some cases, RNA includes any type of RNA (such as one or more of rRNA, tRNA, mRNA, IncRNA, lincRNA, miRNA and / or snRNA). In some cases, RNA includes a mixture of at least one type of RNA. In some cases, DNA can include the following mixture or at least one of them: genomic DNA or nuclear DNA, mitochondrial DNA, Y-series DNA, autosomal DNA, ribosomal DNA or a combination thereof. In some cases, the template is a polymer with any length. In some cases, the template is about 20 bases to about 100 bases, about 30 bases to about 500 bases, about 30 bases to about 1000 bases, about 50 bases to about 300 bases, about 100 bases to about 600 bases, about 200 bases to about 800 bases, about 200 bases to about 600 bases, about 100 bases to about 2000 bases, about 100 bases to about 2500 bases, about 200 bases to about 5000 bases, about 200 bases to about 1000 bases, about 200 to about 10000 bases.In some cases, the template is at least about 10 bases, at least about 20 bases, at least about 30 bases, at least about 40 bases, at least about 50 bases, at least about 60 bases, at least about 70 bases, at least about 80 bases, at least about 90 bases, at least about 100 bases, at least about 150 bases, at least about 200 bases, at least about 250 bases, at least about 300 bases, at least about 350 bases, at least about 400 bases, at least about 450 bases, at least about 500 bases, at least about 550 bases, at least about 600 bases, at least about 650 bases, at least about 700 bases, at least about 750 bases, at least about 800 bases, at least about 900 bases, at least about 100 bases, at least about 150 bases, at least about 200 bases, at least about 250 bases, at least about 300 bases, at least about 350 bases, at least about 400 bases, at least about 450 bases, at least about 500 bases, at least about 550 bases, at least about 600 bases, at least about 650 bases, at least about 700 bases, at least about 750 bases, at least about 8 00 bases, at least about 850 bases, at least about 900 bases, at least about 950 bases, at least about 1000 bases, at least about 1100 bases, at least about 1200 bases, at least about 1300 bases, at least about 1400 bases, at least about 1500 bases, at least about 1700 bases, at least about 2000 bases, at least about 2200 bases, at least about 2500 bases, at least about 2700 bases, at least about 3000, at least about 3500 bases, at least about 4000 bases, at least about 4500 bases, at least about 5000 bases, at least about 10,000 bases or at least about 50,000 bases.In some cases, the template is about or at least about or at most about 10 bases, about or at least about or at most about 20 bases, about or at least about or at most about 30 bases, about or at least about or at most about 40 bases, about or at least about or at most about 50 bases, about or at least about or at most about 60 bases, about or at least about or at most about 70 bases, about or at least about or at most about 80 bases, about or at least about or at most about 90 bases, about or at least about or at most about 100 bases, about or at least about or at most about 150 bases, about or at least about or at most about 160 bases, about or at least about or at most about 170 bases, about or at least about or at most about 180 bases, about or at least about or at most about 190 bases, about or at least about or at most about 200 bases, about or at least about or at most about 210 bases, about or at least about or at most about 220 bases, about or at least about or at most about 230 bases, about or at least about or at most about 240 bases, about or at least about or at most about 250 bases, about or at least about about 200 bases, about or at least about or at most about 250 bases, about or at least about or at most about 300 bases, about or at least about or at most about 350 bases, about or at least about or at most about 400 bases, about or at least about or at most about 450 bases, about or at least about or at most about 500 bases, about or at least about or at most about 550 bases, about or at least about or at most about 600 bases, about or at least about or at most about 650 bases, about or at least about or at most about 700 bases, about or at least about or at most about 750 bases, about or at least about or at most about 800 bases, about or at least about or at most about 850 bases, about or at least about or at most about 900 bases, about or at least about or at most about 950 bases, about or at least about or at most about 1000 bases, about or at least about or at most about 1100 bases, about or at least about or at most about 1200 bases, about or at least about or at most about 1300 bases, about or at least about or at most about 1400 bases, about or at least about or at most about 1500 bases, about or at least about or at most about 1700 bases, about or at least about 1800 bases, about or at least about 1900 bases, about or at least about 2000 bases, about or at least about 2100 bases, about or at least about 2200 bases, about or at least about 2300 bases, about or at least about 2400 bases, about or at least about 2500 bases, about or at least about 2600 bases, about or at least about 2700 bases, about or at least about 2800 bases, about or at least about 2900 bases, about or at least about 3000 bases, about or at least about 3100 bases, about or at least about 3200 bases, about or at least about 3300 bases, about or at least about 3400 bases, about or at least about 3500 bases, about or at least about 3600 bases, about or at least about 3700 bases, about or at least about 3800 bases, about or at least about 3900 bases, about or at least about 4000 bases, about or at least about 4100 bases, about or at least about 4200 bases, about or at least In some cases, the template DNA may be a double-stranded DNA template (dsDNA template) or a single-stranded DNA template (ssDNA template). In some cases, the template RNA may be a double-stranded RNA template (dsRNA template) or a single-stranded RNA template (ssRNA template).

[0214] In some cases, the template is from a single cell. In some cases, the template is from a plurality of cells. In some cases, the template comprises a low copy number DNA or RNA, or a combination of DNA and / or RNA. In some cases, low copy number refers to a sample containing a mixture of equal to or less than about 250 pg (e.g., 100 pg) such as template and / or DNA and / or RNA and / or DNA and RNA. In some cases, RNA may include at least one of: messenger RNA (mRNA), transfer RNA, transfer-messenger RNA, ribosomal RNA, antisense RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), small interfering RNA (siRNA), long non-coding RNA (IncRNA), long insertion non-coding (lincRNA) or any combination thereof. In some cases, the template is from a sample. In some cases, the total amount of template is the total amount of template in the sample. In some cases, the total amount of template is the total amount of template in the reaction mixture. In some cases, the total amount of template is the total amount of template in a pot (e.g., a single container). In some cases, the total amount of template is about 1 femtomolar (fM) to about 100 micromolar, about 40 femtomolar to about 0.01 micromolar, about 50 femtomolar to about 500 femtomolar, about 50 femtomolar to about 0.01 micromolar, about 50 femtomolar to about 0.1 micromolar, about 50 femtomolar to about 500 picomolar, about 50 femtomolar to about 500 nanomolar, about 50 femtomolar to about 500 micromolar, about 50 femtomolar to about 1 picomolar, about 40 femtomolar to about 1 nanomolar, about 1 femtomolar to about 1 picomolar, about 0.0001 micromolar to about 0.01 micromolar, about 0.0001 micromolar to about 0.1 micromolar, or about 0.1 nM to about 100 nM.In some cases, the total amount of template is about equal to or at least about or less than about 1000 micromolar, equal to or at least about or less than about 500 micromolar, equal to or at least about or less than about 250 micromolar, equal to or at least about or less than about 100 micromolar, equal to or at least about or less than about 50 micromolar, equal to or at least about or less than about 25 micromolar, equal to or at least about or less than about 10 micromolar, equal to or at least about or less than about 1 micromolar, equal to or at least about or less than about 0.1 micromolar, equal to or at least about or less than about 0.01 micromolar, equal to or at least about or less than about 0.001 micromolar, equal to or at least about or less than about less than about 0.0001 micromolar, equal to or at least about or less than about 2000 nanomolar, equal to or at least about or less than about 500 nanomolar, equal to or at least about or less than about 250 nanomolar, equal to or at least about or less than about 200 nanomolar, equal to or at least about or less than about 50 nanomolar, equal to or at least about or less than about 25 nanomolar, equal to or at least about or less than about 20 nanomolar, equal to or at least about or less than about 2 nanomolar, equal to or at least about or less than about 0.2 nanomolar, equal to or at least about or less than about 0.01 nanomolar, equal to or at least about or less than about 0.001 nanomolar, equal to or at least about about or less than about 0.0001 nanomoles, equal to or at least about or less than about 3000 picomoles, equal to or at least about or less than about 500 picomoles, equal to or at least about or less than about 250 picomoles, equal to or at least about or less than about 300 picomoles, equal to or at least about or less than about 50 picomoles, equal to or at least about or less than about 25 picomoles, equal to or at least about or less than about 30 picomoles, equal to or at least about or less than about 3 picomoles, equal to or at least about or less than about 0.3 picomoles, equal to or at least about or less than about 0.01 picomoles, equal to or at least about or less than about 0.001 picomoles, equal to or At least about or less than about 0.0001 picomoles, equal to or at least about or less than about 5000 femtomoles, equal to or at least about or less than about 500 femtomoles, equal to or at least about or less than about 250 femtomoles, equal to or at least about or less than about 50 femtomoles, equal to or at least about or less than about 25 femtomoles, equal to or at least about or less than about 10 femtomoles, equal to or at least about or less than about 1 femtomoles, equal to or at least about or less than about 0.1 femtomoles, equal to or at least about or less than about 0.01 femtomoles, equal to or at least about or less than about 0.001 femtomoles, equal to or at least about or less than about 0.0001 femtomoles.

[0215] In some cases, the template may be present in any nucleic acid sample of interest, including but not limited to nucleic acid samples isolated from a single cell, multiple cells (e.g., cultured cells), tissues, organs, or organisms (e.g., bacteria, yeast, etc.). In some cases, nucleic acid samples are isolated from cells, tissues, organs, etc. of mammals (e.g., humans, rodents (e.g., mice), or any other mammal of interest). In some cases, nucleic acid samples are isolated from sources other than mammals, such as bacteria, yeast, insects (e.g., fruit flies), amphibians (e.g., frogs (e.g., African clawed frogs)), viruses, plants, or any other non-mammalian nucleic acid sample sources.

[0216] In some cases, the template is optimized. In some cases, the receptor template or receptor nucleic acid molecule comprises at least one modified nucleotide. In some cases, the receptor template or receptor nucleic acid molecule is engineered to improve template jumping and / or conversion efficiency. In some cases, the receptor template or receptor nucleic acid molecule is optimized at the 3' end. In some cases, optimization prevents secondary structure formation and / or nucleotide composition.

[0217] In some cases, the method disclosed in the present disclosure may further include optimization of the template (e.g., donor template). In some cases, optimization of the template includes contacting the template (e.g., RNA) with a reagent capable of removing the 5' cap structure of the template (e.g., mRNA). In some cases, the removal of the cap structure is performed under conditions that allow the cap structure to be removed by the reagent. In some cases, the method disclosed in the present disclosure further includes, for example, dephosphorylation of the decapping template. In some cases, the method further includes adding a dephosphorylating agent to the decapping template under conditions that allow dephosphorylation.

[0218] In some cases, any method of the present disclosure may further include the optimization of template. In some cases, the optimization of template includes: under the condition of allowing the cap structure to be removed by reagent, the sample containing template is contacted with the reagent of the 5' cap structure of the removal template. In some cases, the optimization of template may further include adding a dephosphorylating agent under the condition of allowing the decapping template to be dephosphorylated by reagent. In some cases, template (such as RNA molecule) is dephosphorylated after synthesis or separation. In some cases, dephosphorylation is realized by treating nucleic acid (such as RNA) molecule with alkaline phosphatase. In some cases, the donor template (such as RNA or mRNA) of separation is decapped and dephosphorylated after separation. Nucleic acid (such as RNA) decapping method includes enzymatic method (such as by using pyrophosphatase, such as tobacco pyrophosphatase) and chemical method (such as periodate oxidation and β elimination). The dephosphorylation method of nucleic acid (such as RNA) can use alkaline phosphatase. In some cases, the mRNA of separation is decapped (such as by using tobacco acid pyrophosphatase) and dephosphorylation (such as by using alkaline phosphatase). In some cases, the RNA cap structure is removed by enzymatic treatment of the mRNA with pyrophosphatase or by chemical decapping (e.g., by periodate oxidation and beta elimination). In some cases, the mRNA is modified with a tag.

[0219] In some cases, template hopping depends on the concentration of the acceptor nucleic acid molecule.

[0220] In some cases, the method of the present disclosure further comprises reverse transcription of the template nucleic acid molecule using a modified reverse transcriptase to obtain a nucleic acid molecule. In some cases, the nucleic acid molecule is a free nucleic acid molecule. In some cases, the template nucleic acid molecule is a free nucleic acid molecule.

[0221] In some cases, amplification products are generated using primer extension or stretching reactions. Primer extension / stretching reactions can include cycles of incubating the reaction mixture at a denaturing temperature for a denaturing duration and incubating the reaction mixture at a stretching temperature for a stretching duration.

[0222] Any type of nucleic acid amplification reaction can be used to amplify target nucleic acid and produce amplified products. In addition, the amplification of nucleic acid can be linear, exponential or a combination thereof. Amplification can be based on emulsion or can be not based on emulsion. Non-limiting examples of nucleic acid amplification methods include reverse transcription, primer extension, polymerase chain reaction, ligase chain reaction, helicase-dependent amplification, asymmetric amplification, rolling circle amplification and multiple displacement amplification (MDA). In some cases, the amplified product can be DNA. In the case of amplified target RNA, DNA can be obtained by reverse transcription RNA, and the subsequent amplification of DNA can be used to produce amplified DNA products. Amplified DNA products can indicate the presence of target RNA in biological samples. In the case of amplified DNA, any DNA amplification can be adopted. Non-limiting examples of DNA amplification methods include polymerase chain reaction (PCR), variants of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetric staggered PCR, decremental PCR), and ligase chain reaction (LCR). In some cases, DNA amplification is linear. In some cases, DNA amplification is exponential. In some cases, DNA amplification is achieved using nested PCR, which can improve the sensitivity of detecting amplified DNA products.

[0223] Denaturation temperature can vary depending on, for example, the specific biological sample analyzed, the specific source of the target nucleic acid in the biological sample (e.g., virions, bacteria), the reagents used, and / or the required reaction conditions. In some cases, the denaturation temperature can be about 80°C to about 110°C. In some cases, the denaturation temperature can be about 90°C to about 100°C. In some cases, the denaturation temperature can be about 90°C to about 97°C. In some instances, the denaturation temperature can be about 92°C to about 95°C. In other instances, the denaturation temperature can be about 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C.

[0224] The denaturation duration may vary depending on, for example, the specific biological sample analyzed, the specific source of the target nucleic acid in the biological sample (e.g., virions, bacteria), the reagents used, and / or the required reaction conditions. In some cases, the denaturation duration may be less than or equal to about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, the denaturation duration may not exceed about 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

[0225] The stretching or extension temperature may vary depending on, for example, the specific biological sample being analyzed, the specific source of the target nucleic acid in the biological sample (e.g., viral particles, bacteria), the reagents used, and / or the desired reaction conditions. In some cases, the stretching temperature may be about 30°C to about 80°C. In some cases, the stretching temperature may be about 35°C to about 72°C. In some cases, the stretching temperature may be about 45°C to about 68°C. In some cases, the stretching temperature may be about 35°C to about 65°C. In some cases, the stretching temperature may be about 40°C to about 67°C. In some cases, the stretching temperature may be about 50°C to about 68°C. In some cases, the stretching temperature can be about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 34°C, 33°C, 32°C, 31°C, 30°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 35°C, 36°C, 37°C, 38°C, 39°C. , 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C or 80°C.

[0226] The stretching duration can vary depending on the specific biological sample, the specific source (e.g., viral particles, bacteria) of the target nucleic acid in the biological sample, the reagent used and / or the required reaction conditions, for example, analyzed. In some cases, the stretching duration can be less than or equal to about 360 seconds, less than or equal to about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds or 1 second. In some cases, the stretching duration may be no more than about 120 seconds, 90 seconds, 80 seconds, 70 seconds, 65 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds or 1 second.

[0227] In some cases, multiple cycles of primer extension reactions can be performed. Any suitable number of cycles can be performed. In some cases, the number of cycles performed may be less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 cycles. The number of cycles performed may depend on, for example, the number of cycles (e.g., cycle threshold (Ct)) required for obtaining a detectable amplified product (e.g., indicating the presence of a detectable amount of amplified DNA product of target RNA in a biological sample). In some cases, the number of cycles required for obtaining a detectable amplified product (e.g., indicating the presence of a detectable amount of a DNA product of target RNA in a biological sample) may be less than about or about 100 cycles, 75 cycles, 70 cycles, 65 cycles, 60 cycles, 55 cycles, 50 cycles, 40 cycles, 35 cycles, 30 cycles, 25 cycles, 20 cycles, 15 cycles, 10 cycles, 8 cycles, 7 cycles, 5 cycles or 4 cycles. In addition, in some cases, a detectable amount of amplifiable product (e.g., a detectable amount of DNA product indicating the presence of target RNA in a biological sample) can be obtained at a cycle threshold (Ct) of less than 100, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.

[0228] In some cases, the amplification step (e.g., primer amplification, template amplification, nucleic acid amplification) comprises a PCR step. In some cases, each PCR cycle may comprise a denaturation step, an annealing step, and an extension step. In some cases, each PCR cycle may comprise a denaturation step and an extension step. In some cases, PCR comprises at least about or about or at most about 1 cycle, at least about or about or at most about 4 cycles, at least about or about or at most about 5 cycles, at least about or about or at most about 10 cycles, at least about or about or at most about 15 cycles, at least about or about or at most about 20 cycles, at least about or about or at most about 25 cycles, at least about or about or at most about 30 cycles, at least about or about or at most about 35 cycles, at least about or about or at most about 40 cycles, at least about or about or at most about 45 cycles, at least about or about or at most about 50 cycles, at least about or about or at most about 55 cycles, at least about or about or at most about 56 cycles, at least about or about or at most about 58 cycles, at least about or about or at most about 59 cycles, at least about or about or at most about 60 cycles, at least about or about or at most about 61 cycles, at least about or about or at most about 62 cycles, at least about or about or at most about 64 cycles, at least about or about or at most about 65 cycles, at least about or about or at most about 66 cycles, at least about or about At most about 60 cycles, at least about or about or at most about 65 cycles, at least about or about or at most about 70 cycles, at least about or about or at most about 75 cycles, at least about or about or at most about 80 cycles, at least about or about or at most about 90 cycles, at least about or about or at most about 95 cycles, at least about or about or at most about 100 cycles, at least about or about or at most about 110 cycles, at least about or about or at most about 120 cycles, at least about or about or at most about 130 cycles, at least about or about or at most about 140 cycles, at least about or about or at most about 150 cycles, at least about or about or at most about 160 cycles. In some cases, PCR comprises about 10 cycles to 40 cycles, about 20 cycles to 40 cycles, about 20 cycles to 38 cycles, about 20 cycles to 35 cycles, about 10 cycles to 35 cycles, about 10 cycles to 30 cycles, about 25 cycles to 30 cycles, about 20 cycles to 30 cycles, about 4 cycles to 8 cycles, or about 28 cycles to 32 cycles. In some cases, the reaction is heated to 95°C for 3 minutes before the PCR cycle begins. In some cases, each PCR cycle comprises holding 95°C for 3 seconds and holding 62°C for 20 seconds. In some cases, each PCR cycle comprises holding 95°C for 3 seconds, holding 54°C for 10 seconds, and holding 64°C for 20 seconds. In some cases, each PCR cycle comprises holding 95°C for 3 seconds and holding 64°C for 20 seconds. In some cases, each PCR cycle comprises holding 95°C for 3 seconds and holding 64°C for 20 seconds. In some cases, each PCR cycle comprises holding 95°C for 3 seconds and holding 62°C for 60 seconds. In some cases, each PCR cycle comprises holding 95° C. for 3 seconds, holding 54° C. for 10 seconds, and holding 64° C. for 10 seconds. In some cases, the PCR comprises 30 cycles. In some cases, the reaction is heated to 68° C. after the PCR cycles are completed. In some cases, the reaction is heated to 68° C. after the PCR cycles are completed for about 1 second to about 5 seconds, about 1 second to about 5 minutes, about 1 minute to about 5 minutes.In some cases, the PCR methods described herein comprise an extension or stretching step that is at least about 5 seconds long, at least about 6 seconds long, at least about 7 seconds long, at least about 8 seconds long, at least about 9 seconds long, at least about 10 seconds long, at least about 11 seconds long, at least about 12 seconds long, at least about 13 seconds long, at least about 14 seconds long, at least about 15 seconds long, at least about 20 seconds long, at least about 30 seconds long, at least about 40 seconds long, at least about 50 seconds long, at least about 60 seconds long, at least about 90 seconds long, at least about 120 seconds long, at least about 150 seconds long, at least about 180 seconds long, at least about 210 seconds long, at least about 240 seconds long, at least about 270 seconds long, at least about 300 seconds long, at least about 330 seconds long, at least about 360 seconds long, at least about 390 seconds long, or longer.

[0229] The time for amplification to obtain a detectable amount of amplification product indicating the presence of the amplified target nucleic acid can vary depending on the biological sample from which the target nucleic acid is obtained, the specific nucleic acid amplification reaction to be performed, and the specific number of cycles of the desired amplification reaction. In some cases, amplification of the target nucleic acid can obtain a detectable amount of amplification product indicating the presence of the target nucleic acid within the following time periods: 120 minutes or less; 90 minutes or less; 60 minutes or less; 50 minutes or less; 45 minutes or less; 40 minutes or less; 35 minutes or less; 30 minutes or less; 25 minutes or less; 20 minutes or less; 15 minutes or less; 10 minutes or less; or 5 minutes or less.

[0230] In some cases, the biological sample can be preheated before performing the primer extension reaction. The temperature (e.g., preheating temperature) and duration (e.g., preheating duration) of the preheated biological sample can vary depending on, for example, the specific biological sample analyzed. In some instances, the biological sample can be preheated for no more than about 60 minutes, 50 minutes, 40 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or 5 seconds. In some instances, the biological sample can be preheated at a temperature of about 80°C to about 110°C. In some instances, the biological sample can be preheated at a temperature of about 90°C to about 100°C. In some instances, the biological sample can be preheated at a temperature of about 90°C to about 97°C. In some instances, the biological sample can be preheated at a temperature of about 92°C to about 95°C. In some cases, the biological sample can be preheated at a temperature of about 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C or 100°C.

[0231] In some cases, the reagents required for nucleic acid amplification may also include a reporter that obtains a detectable signal, and the presence or absence of the detectable signal indicates the presence of the amplified product. The intensity of the detectable signal may be proportional to the amount of the amplified product. In some cases of amplified products of nucleic acids different from the target nucleic acid type initially amplified, the intensity of the detectable signal may be proportional to the amount of the target nucleic acid initially amplified. For example, in the case of amplifying target RNA by parallel reverse transcription and amplifying DNA obtained from reverse transcription, the reagents required for the two reactions may also include a reporter that can obtain a detectable signal indicating the presence of the amplified DNA product and / or the amplified target RNA. The intensity of the detectable signal may be proportional to the amount of the amplified DNA product and / or the amplified original target RNA. The use of a reporter also realizes real-time amplification methods, including real-time PCR for DNA amplification.

[0232] The reporter can be connected to the nucleic acid (including the amplified product) by covalent or non-covalent connection or interaction. Non-limiting examples of non-covalent connection or interaction include ionic interaction, van der Waals force, hydrophobic interaction, hydrogen bonding and combinations thereof. In some cases, the reporter can be combined with the initial reactant, and the reporter content change can be used to detect the amplified product. In some cases, the reporter may only be detectable (or undetectable) when nucleic acid amplification is carried out. In some cases, optically active dyes (such as fluorescent dyes) can be used as reporters. Non-limiting examples of dyes include SYBR green, SYBR blue, DAPI, propidium iodide, Hoeste, SYBR gold, ethidium bromide, acridine, proflavine, acridine orange, acridine yellow, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyl, anthramycin, phenanthridine, and acridine, ethidium bromide, propidium iodide, hexidium iodide, iodide), dihydroethidium, ethidium homodimer-1 and ethidium homodimer-2, ethidium monoazide and ACMA, Hoechst 33258, Hoechst 33342, Hoechst34580, DAPI, acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOXOrange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-P RO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBRDX, SYTO-40, SYTO-41, SYTO-42, SYTO-43, SYTO-44, SYTO-45 (blue), SYTO-13, SYTO-16, SYTO-24, SYTO-21, SYTO-23, SYTO-12, SYTO-11, SYTO-20, SYTO-22, SYTO-15, SYTO-14, SY TO-25 (green), SYTO-81, SYTO-80, SYTO-82, SYTO-83, SYTO-84, SYTO-85 (orange), SYTO-64, SYTO-17, SYTO-59, SYTO-61, SYTO-62, SYTO-60, SYTO-63 (red), fluorescein, fluorescein isothiocyanate isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II, ethidium homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosin, coumarin, methylcoumarin, pyrene, malachite green, stilbene, lucifer yellow, cascade blue, blue), dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes (e.g., including europium and terbium complexes), carboxytetrachlorofluorescein, 5-carboxy and / or 6-carboxyfluorescein (FAM), 5-(or 6-)iodoacetamidofluorescein, 5-{[2(and 3)-5-(acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein), lissamine rhodamine B sulfonyl chloride, 5 and / or 6 carboxyrhodamine (ROX), 7-amino-methyl-coumarin, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophore, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, 3,6-disulfonate-4-amino-naphthalimide, phycobiliprotein, AlexaFluor 350, AlexaFluor550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 755, and DyLight 800 dyes, or other fluorophores.

[0233] In some cases, the reporter can be a sequence-specific oligonucleotide probe that is optically active when hybridized with the amplified product. Since the probe is specifically combined with the amplified product sequence, specificity and detection sensitivity can be increased using the oligonucleotide probe. The probe can be connected to any optically active reporter (e.g., dye) and can also include a quencher that can block the optical activity of the associated dye. Non-limiting examples of probes that may be suitable for use as reporter include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes. In some cases, the reporter can be a radioactive species. Non-limiting examples of radioactive species include 14C, 1231, 1241, 1251, 1311, 99mTc, 35S, or 3H. In some cases, the reporter can be an enzyme that can produce a detectable signal. The detectable signal can be produced by the activity of the enzyme using its substrate or using a specific substrate when the enzyme has multiple substrates. Non-limiting examples of enzymes that can be used as reporters include alkaline phosphatase, horseradish peroxidase, 12-galactosidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, and luciferase.

[0234] In some cases, the amplified product (e.g., amplified DNA product, amplified RNA product) can be detected. The detection of the amplified product (including amplified DNA) can be achieved using any suitable detection method. The specific type of the detection method used can depend on, for example, the specific amplified product, the type of reaction vessel used for amplification, other reagents in the reaction mixture, whether a reporter is included in the reaction mixture, and if a reporter is used, the specific type of the reporter used. Non-limiting examples of detection methods include optical detection, spectral detection, electrostatic detection, electrochemical detection, etc. Optical detection methods include, but are not limited to, fluorescence determination and UV-Vis light absorption. Spectral detection methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy. Electrostatic detection methods include, but are not limited to, gel-based techniques, such as gel electrophoresis, SDS-PAGE gel. Electrochemical detection methods include, but are not limited to, electrochemical detection of the amplified product after high performance liquid chromatography separation of the amplified product.

[0235] In some cases, the time required for the key element of the method of completion can vary depending on the specific steps of the method. In some cases, the amount of time for the key element of the method of completion can be about 5 minutes to about 120 minutes. In some cases, the amount of time for the key element of the method of completion can be about 5 minutes to about 60 minutes. In some cases, the amount of time for the key element of the method of completion can be about 5 minutes to about 30 minutes. In some cases, the amount of time for the key element of the method of completion can be less than or equal to 120 minutes, less than or equal to 90 minutes, less than or equal to 75 minutes, less than or equal to 60 minutes, less than or equal to 45 minutes, less than or equal to 40 minutes, less than or equal to 35 minutes, less than or equal to 30 minutes, less than or equal to 25 minutes, less than or equal to 20 minutes, less than or equal to 15 minutes, less than or equal to 10 minutes or less than or equal to 5 minutes.

[0236] In some cases, the reaction can have a pH suitable for producing a product, suitable for primer extension, protein expression, PCR amplification, or template hopping. In some cases, the pH of the reaction can range from about 5 to about 9, about 6 to about 9, about 7 to about 9, about 8 to about 9. In some cases, the pH range is about pH 2 to about pH 10, about pH 4 to about pH 10, about pH 2 to about pH 8, about pH 4 to about pH 8, about pH 5 to about pH 8, about pH 5 to about pH 7, about pH 6 to about pH 11, about pH 6 to about pH 12, about pH 5 to pH 13, about pH 5 to about pH 14. In some cases, the pH is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14.

[0237] In some cases, any of the methods of the present disclosure can include a detergent. In some cases, the detergent is a nonionic and / or zwitterionic detergent. In some cases, the nonionic detergent is selected from the group consisting of tween, triton, Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-SM, Triton N-101 (polyoxyethylene branched nonylphenyl ether), Triton QS-15, Triton QS-44, Triton RW-75 (polyethylene glycol 260 mono (hexadecyl / octadecyl) ether and 1-octadecyl alcohol), Triton X-100 (polyethylene glycol tert-octylphenyl ether), Triton X-102, Triton X-15, Triton X-151, Triton X-200, Triton X-207, Triton X-114, Triton X-165, Triton X-305, Triton X-405 (polyoxyethylene (40) isooctylphenyl ether), reduced Triton X-405 (polyoxyethylene (40) isooctyl cyclohexyl ether), Triton X-45 (polyoxyethylene glycol 4-tert-octylphenyl ether), Triton X-705-70, any form of TWEEN (including: TWEEN 20 (polyoxyethylene sorbitan monolaurate), TWEEN 21 (polyoxyethylene sorbitan monolaurate), TWEEN 40 (polyoxyethylene (20) sorbitan monopalmitate), TWEEN 60 (polyoxyethylene glycol sorbitan monostearate), TWEEN 61 (polyoxyethylene glycol sorbitan monostearate), TWEEN 65 (polyoxyethylene sorbitan tristearate), TWEEN 80 (polyoxyethylene sorbitan monooleate), TWEEN 81 (polyoxyethylene sorbitan monooleate), TWEEN 85 (polyoxyethylene (20) sorbitan trioleate)), Brij, Brij Brij 30 (polyoxyethylene 4 lauryl ether), Brij 35 (polyoxyethylene 23 lauryl ether), Brij 52 (polyoxyethylene 2 cetyl ether), Brij 56 (polyoxyethylene 10 cetyl ether), Brij 58 (polyoxyethylene 20 cetyl ether), Brij 72 (polyoxyethylene 2 stearyl ether), Brij 76 (polyoxyethylene 10 stearyl ether), Brij 78 (polyoxyethylene 20 stearyl ether), Brij 92 (polyoxyethylene 2 oleyl ether), Brij 97 (polyoxyethylene 10 oleyl ether), Brij 98 (polyoxyethylene 20 oleyl ether), Brij 700 (polyoxyethylene 100 stearyl ether), octylthioglucoside, maltoside and combinations thereof.

[0238] In some cases, any method for producing any molecule disclosed herein includes at least one salt. In some cases, salt is selected from at least one member of the group consisting of: NaCl, LiCl, AlCl3, CuCl2, MgCl2, InCl3, SnCl4, CrCl2, CrCl3, KCl, NaI, KI, TMACl (tetramethylammonium chloride), TEACl (tetraethylammonium chloride), KSCN, CsSCN, KCH3COO, CH3COONa, C5H8KNO4, C5H8NNaO4, CsCl and any combination thereof. In some cases, any method for producing any molecule disclosed herein includes NaCl. In some cases, the condition enough to produce molecule or library includes NaCl. In some cases, reaction can have salt concentration and / or NaCl concentration suitable for producing product, suitable for primer extension, protein expression, pcr amplification or template jumping. In some cases, the NaCl concentration is about 50 mM to about 1000 mM, about 100 mM to about 500 mM, about 200 mM to about 300 mM, about 200 mM to about 600 mM. In some cases, the NaCl concentration is at least about, at most about, or about 50 mM, at least about, at most about, or about 100 mM, at least about, at most about, or about 150 mM, at least about, at most about, or about 200 mM, at least about, at most about, or about 250 mM, at least about, at most about, or about 300 mM, at least about, at most about, or about 350 mM, at least about, at most about, or about 400 mM, at least about, at most about, or about 450 mM, at least about, at most about, or about 500 mM. In some cases, NaCl can improve the enzyme activity and / or template hopping of an enzyme or polypeptide (e.g., reverse transcriptase) of the present invention.

[0239] In some cases, the reaction can have a nucleotide (e.g., dNTP) concentration suitable for producing a product, suitable for primer extension, protein expression, PCR amplification, or template hopping. In some cases, the total dNTP concentration in the reaction can be about 50 μM to about 1000 μM, about 100 μM to about 500 μM, about 200 μM to about 300 μM, about 200 μM to about 600 μM. In some cases, the total dNTP concentration is at least about, at most about, or about 50 μM, at least about, at most about, or about 100 μM, at least about, at most about, or about 150 μM, at least about, at most about, or about 200 μM, at least about, at most about, or about 250 μM, at least about, at most about, or about 300 μM, at least about, at most about, or about 350 μM, at least about, at most about, or about 400 μM, at least about, at most about, or about 450 μM, at least about, at most about, or about 500 μM. M, at least about, at most about, or about 550 μM, at least about, at most about, or at least about, at most about, or about 600 μM, at least about, at most about, or about 650 μM, at least about, at most about, or about 700 μM, at least about, at most about, or about 750 μM, at least about, at most about, or about 800 μM, at least about, at most about, or about 850 μM, at least about, at most about, or about 900 μM, at least about, at most about, or about 950 μM, or at least about, at most about, or about 1000 μM. In some cases, the total concentration of each dNTP is at least about, at most about, or about 1 μM; at least about, at most about, or about 2 μM; at least about, at most about, or about 3 μM; at least about, at most about, or about 4 μM; at least about, at most about, or about 5 μM; at least about, at most about, or about 6 μM; at least about, at most about, or about 7 μM; at least about, at most about, or about 8 μM; at least about, at most about, or about 9 μM; at least about, at most about, or about 10 μM; at least about, at most about, or about 15 μM; at least about, at most about, or about 20 μM; at least about, at most about, or about 25 μM; at least about, at most about, or about 30 μM; at least about, at most about, or about 35 μM; at least about, at most about, or about 40 μM ; at least about, at most about, or about 45 μM; at least about, at most about, or about 50 μM; at least about, at most about, or about 55 μM; at least about, at most about, or about 60 μM; at least about, at most about, or about 65 μM; at least about, at most about, or about 70 μM; at least about, at most about, or about 75 μM; at least about, at most about, or about 80 μM; at least about, at most about, or about 85 μM; at least about, at most about, or about 90 μM; at least about, at most about, or about 95 μM; at least about, at most about, or about 100 μM; at least about, at most about, or about 250 μM; at least about, at most about, or about 500 μM; at least about, at most about, or about 1000 μM; at least about, at most about, or about 10000 μM.In some cases, the total concentration of each dNTP is about 2 μM to about 5 μM, about 2 μM to about 10 μM, about 2 μM to about 20 μM, about 2 μM to about 50 μM, about 2 μM to about 100 μM, about 2 μM to about 250 μM, about 5 μM to about 10 μM, about 5 μM to about 50 μM, about 5 μM to about 250 μM, about 5 μM to about 1000 μM.

[0240] In some cases, the concentration of each dNTP may be independent of and different from the concentration of one or more dNTPs. In some cases, the concentration of each dNTP (e.g., the concentration of each of dCTP, dGTP, dTTP or dATP) may be independent of and different from the concentration of at least one other dNTP. In some cases, the concentration of a dNTP (e.g., dCTP, dGTP, dTTP or dATP) differs from the concentration of at least one other dNTP (e.g., dCTP, dGTP, dTTP or dATP) by at least about or at most about or about 1 times, 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 20 times, 35 times, 50 times, 75 times, 90 times, 100 times, 200 times, 500 times or 1000 times.

[0241] In some cases, the reaction mixture includes a pH adjusting agent. The pH adjusting agents of interest include, but are not limited to, sodium hydroxide, hydrochloric acid, phosphate buffer solution, tris buffer, citric acid buffer solution, etc. For example, the pH value of the reaction mixture can be adjusted to a desired range by adding an appropriate amount of a pH adjusting agent.

[0242] The temperature range suitable for producing the product can vary depending on factors such as the specific polymerase employed, the melting temperature of any optional primers employed, etc. In some cases, the polymerase can include, but is not limited to, a reverse transcriptase, a Moloney murine leukemia virus (MMLV) reverse transcriptase, an R2 reverse transcriptase, an RNA-guided DNA polymerase, a DNA-guided DNA polymerase, a non-LTR retrotransposon, an R2 non-LTR retrotransposon, a polypeptide having reverse transcriptase activity, or any variant thereof, or any combination thereof.

[0243] In some cases, conditions sufficient to produce a product include bringing the reaction mixture to a temperature in the range of about 4°C to about 72°C, about 16°C to about 70°C, about 37°C to about 50°C, about 40°C to about 45°C, about 30°C to about 42°C, about 25°C to about 42°C, about 25°C to about 30°C, about 28°C to about 32°C, about 29°C to about 31°C. In some cases, the temperature is about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C In some cases, the temperature is about or up to about 42°C. In some cases, the temperature is about or up to about 50°C. In some cases, the temperature is about or up to about 35°C. In some cases, the temperature is about or up to about 25°C. In some cases, the temperature is about or up to about 30°C. In some cases, the reaction is cultivated for about 20 minutes to about 3 hours, about 30 minutes to about 1.5 hours, about 30 minutes to about 1 hour, about 30 minutes to about 2 hours, about 1 hour to about 2 hours, about 1 hour to about 1.5 hours, about 30 minutes to about 5 hours, about 1 hour to about 3 hours, about 1 hour to about 4 hours, about 1 hour to about 5 hours. In some cases, the reaction is cultivated for about 1 hour. In some cases, the reaction is cultivated for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours or about 5 hours. In some cases, the reaction is cultivated for at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 1 hour, at least about 1.5 hours, at least about 2 hours, at least about 2.5 hours, at least about 3 hours, at least about 3.5 hours, at least about 4 hours, at least about 4.5 hours or at least about 5 hours. In some cases, the reaction is cultivated for about 1 hour at about 30°C or cultivated for about 1 hour at about 42°C. In some cases, the conditions sufficient to produce molecules or nucleic acid molecules include a temperature of about 12°C to about 42°C for about 1 minute to about 5 hours. In some cases, the conditions sufficient to produce molecules or nucleic acid molecules include a temperature of about 8°C to about 50°C for about 1 minute to about 24 hours.

[0244] In some cases, primer can be designed to a certain length. In some cases, the length of primer can be about 6 to about 100 nucleotides, about 6 to about 90 nucleotides, about 6 to about 80 nucleotides, about 6 to about 70 nucleotides, about 6 to about 60 nucleotides, about 6 to about 50 nucleotides, about 6 to about 40 nucleotides, about 6 to about 30 nucleotides, about 6 to about 20 nucleotides or about 6 to about 10 nucleotides. In some cases, the length of primer can be about 25 to about 80, about 25 to about 75, about 25 to about 70, about 25 to about 65, about 25 to about 60, about 25 to about 55, about 25 to about 50, about 25 to about 45, about 25 to about 40, about 25 to about 35 or about 25 to about 30 bases. In some cases, a primer can be at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases in length. In some cases, the length of a primer can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 bases. In some cases, the primers can be at least about, at most about, or about 120, 130, 140, 150, 160, 170, 180, 190, 200, 230, 250, 270, 290, 300, 320, 340, 350, 370, 400, 420, 450, 470, 490, or 500.

[0245] In some cases, primers can be designed to anneal to a target at a given melting temperature (Tm). In some cases, Tm can be about 20°C to about 100°C, about 20°C to about 90°C, about 20°C to about 80°C, about 20°C to about 70°C, about 20°C to about 60°C, about 20°C to about 50°C, about 20°C to about 40°C, or about 20°C to about 30°C. In some cases, the Tm can be at least about, at most about, or about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 83 ℃, 84 ℃, 85 ℃, 96 ℃, 97 ℃, 98 ℃, 99 ℃ or 100 ℃. The multiple primers can be designed to have a Tm within the following range, for example, within a range spanning: 15 ℃, 10 ℃, 9 ℃, 8 ℃, 7 ℃, 6 ℃, 5 ℃, 4 ℃, 3 ℃, 2 ℃ or 1 ℃. The multiple primers can be designed to have a consistent Tm.

[0246] In some cases, enzymes of the present disclosure, or modified enzymes (e.g., modified reverse transcriptases), or proteins, or polypeptides, or variants or PCR products, or cDNA molecules, or templates, or nucleic acid molecules, or any components can be purified. In some cases, fragmented or degraded nucleic acids (e.g., RNA or DNA) can be purified. In some cases, reverse transcriptases or modified reverse transcriptases can be purified. In some cases, R2 reverse transcriptases or modified R2 reverse transcriptases can be purified. In some cases, non-LTR retrotransposon proteins or polypeptides with reverse transcriptase activity, or modified non-LTR retrotransposon proteins or modified polypeptides with reverse transcriptase activity can be further purified. In some cases, cDNA molecules can be purified. In some cases, templates can be purified. In some cases, receptor nucleic acid molecules can be purified.

[0247] Purification can include precipitation, ultracentrifugation, chromatography based on size, charge, hydrophobicity, affinity, metal binding, HPLC. In some cases, purification includes column chromatography. In some cases, column chromatography can be size exclusion (SEC), ion exchange (IEX), affinity chromatography, immobilized metal ion affinity chromatography (IMAC), Ni-IMAC chromatography and / or hydrophobic interaction (HIC). In some cases, purification includes His tag affinity resin. In some cases, purification can include one step. In some cases, purification can include two steps. In some cases, two-step purification includes nickel and heparin. In some cases, two-step purification includes nickel affinity purification and heparin affinity purification. In some cases, compared to one-step purification, two purification steps provide higher activity and / or increased template hopping. In some cases, purification includes heparin affinity purification. In some cases, purification can include affinity purification, Ni-NTA affinity purification, fast protein liquid chromatography (FPLC) (e.g., AKTA FPLC system and Bio-Rad FPLC system), high pressure liquid chromatography (HPLC) (e.g., Beckman HPLC and Waters HPLC). In some cases, purification can include, but is not limited to, ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradient, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reverse phase purification, ceramic HYPERD (registered trademark) ion exchange chromatography, and hydrophobic interaction column (HIC). Also included are analytical methods, such as SDS-PAGE (e.g., Coomassie, silver staining), immunoblotting, Bradford, and ELISA, which can be utilized during any step of the production or purification process, and are typically used to measure the purity of a protein or enzyme composition.

[0248] In some cases, the overall activity of the enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein or polypeptide with reverse transcriptase activity, reverse transcriptase, or variant thereof, or product thereof purified using the two-step purification is at least about 2%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% higher than the overall activity using a single-step purification. In some cases, the overall activity of the purified enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein or polypeptide with reverse transcriptase activity, reverse transcriptase, or variants thereof, or its product is at least about 0.5%, at least about 1%, at least about 1.5%, or more than the overall activity of the unpurified enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein or polypeptide with reverse transcriptase activity, reverse transcriptase, or variants thereof, or its product. At least about 5%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%.In some cases, the purified enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein, or polypeptide having reverse transcriptase activity, reverse transcriptase, modified enzyme, modified reverse transcriptase, modified polypeptide having reverse transcriptase activity, or variants thereof, or products thereof have at least about 0.5%, at least about 1%, at least about 3%, at least about or about 5%, at least about or about 10%, at least about or about 15%, at least about or about 20%, at least about or about 25%, at least about or about 30%, at least about or about 35%, at least about or about 40%, at least about or about 45%, at least about or about 50%, at least about or about 55%, at least about or about 60%, at least about or about 61%, at least about or about 62%, at least about or about 63%, at least about or about 64%, at least about or about 65%, at least about or about 66%, at least about or about 67%. %, at least about or about 68%, at least about or about 69%, at least about or about 70%, at least about or about 71%, at least about or about 72%, at least about or about 73%, at least about or about 74%, at least about or about 75%, at least about or about 76%, at least about or about 77%, at least about or about 78%, at least about or about 79%, at least about or about 80%, at least about or about 81%, at least about or about 82%, at least about or about 83%, At least about or about 84%, at least about or about 85%, at least about or about 86%, at least about or about 87%, at least about or about 88%, at least about or about 89%, at least about or about 90%, at least about or about 91%, at least about or about 92%, at least about or about 93%, at least about or about 94%, at least about or about 95%, at least about or about 96%, at least about or about 97%, at least about or about 98%, or at least about or about 99% pure.

[0249] In some cases, the intensity of template hopping produced by the purified enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein or polypeptide with reverse transcriptase activity, reverse transcriptase, or variants thereof, or its product is more and / or higher than that of the unpurified enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein or polypeptide with reverse transcriptase activity, reverse transcriptase, or variants thereof, or its product by at least about or about one time, at least about or about two times, at least about or about three times, at least about or about four times, at least about or about five times, at least about or about six times, at least about or about seven times, at least about or about eight times, at least about or about eight times, at least about or about ten ... At least about or about nine times, at least about or about ten times, at least about or about fifteen times, at least about or about twenty times, at least about or about twenty-five times, at least about or about thirty times, at least about or about forty times, at least about or about fifty times, at least about or about seventy times, at least about or about eighty times, at least about or about ninety times, at least about or about 100 times, at least about or about 150 times, at least about or about 200 times, at least about or about 250 times, at least about or about 300 times, at least about or about 350 times, at least about or about 400 times, at least about or about 500 times, at least about or about 700 times, at least about or about 1000 times, at least about or about 10,000 times.

[0250] Enzyme mutation

[0251] In some cases, modified enzymes or derivatives and variants can be prepared during peptide synthesis or by post-production modification. In some cases, modified enzymes or derivatives and variants can be prepared by site-directed mutagenesis (e.g. Site-directed mutagenesis kit protocol), random mutagenesis or enzymatic cleavage and / or nucleic acid ligation. In some cases, derivatives and variants or modified enzymes are produced by random mutagenesis. In some cases, rational design and / or mutagenesis are based on sequence alignment analysis. In some cases, rational design / mutagenesis is based on sequence alignment analysis of determined and known enzymes and proteins. In some cases, sequence alignment analysis or homology simulation is performed on the homology of enzymes and / or elements to R2, such enzymes and / or elements include but are not limited to non-LTR retrotransposons, telomerase, II group introns, LTR retrotransposons, reverse transcriptases, retroviral reverse transcriptases (e.g., HIV, MMLV) and viral RNA dependent RNA polymerases.

[0252] In some cases, variants or modified enzymes of the present disclosure can be produced by, for example, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis and directed evolution, as well as various other recombination methods. Methods for preparing modified enzymes, polynucleotides and proteins (e.g., variants) include: DNA shuffling methods; methods based on non-homologous recombination of genes, such as ITCHY (see Ostermeier et al., 7:2139-44

[1999] ), SCRACHY (see Lutz et al. 98:11248-53

[2001] ), SHIPREC (see Sieber et al. 19:456-60

[2001] ) and NRR (see Bittke et al., 19:456-60

[2001] ). r et al., 20:1024-9

[2001] ; Bittker et al., 101:7011-6

[2004] ); and methods that rely on the use of oligonucleotides to insert random and targeted mutations, deletions and / or insertions (see Ness et al., 20:1251-5

[2002] ; Coco et al., 20:1246-50

[2002] ; Zha et al., 4:34-9

[2003] ; Glaser et al., 149:3903-13

[1992] ). In some cases, the polynucleotides, polypeptides, proteins or enzymes of the present disclosure may be altered by random mutagenesis by means of error-prone PCR, random nucleotide insertion or other methods prior to recombination. The polynucleotides, polypeptides, proteins or enzymes of the present disclosure may be produced by DNA shuffling, gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling involves assembling two or more DNA segments by homologous or site-directed recombination to produce polynucleotide sequence changes. DNA shuffling can be used to modulate the activity of the polynucleotides, polypeptides, proteins or enzymes of the present disclosure, and these methods can be used to generate polypeptides with altered activity.See generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; 5,837,458; and 6,444,468; and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998). The polynucleotides, polypeptides, proteins or enzymes of the present disclosure may contain one or more components, motifs, segments, parts, domains, fragments, etc. of the polynucleotides, polypeptides, proteins or enzymes of the present disclosure. In some cases, a kit for mutational PCR may be used, such as the Diversify PCR random mutagenesis kit (Clontech) or the GeneMorph random mutagenesis kit (Stratagene).

[0253] In some cases, variant protein and parent protein or modified enzyme and wild-type or unmodified enzyme and each other are different in a few amino acid residues.The number of different amino acid residues can be one or more, preferably about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues.In some cases, the number of different amino acids between variants is between about 1 and about 10.In some cases, related protein and especially variant protein comprise at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% amino acid sequence identity.In addition, related protein or variant protein as used herein refers to the protein that the number of significant regions is different from another related protein or parent protein. For example, in some cases, the variant protein has about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 corresponding significant regions that differ from the parent protein.

[0254] In some cases, the screening method may include conventional screening methods, such as liquid phase analysis or microtiter plate-based analysis. The format for liquid phase analysis is usually a 96-well, 384-well or 1536-well microtiter plate manipulated by a robot. Other screening methods include growth selection (Snustad et al., 1988; Lundberg et al., 1993; Yano et al., 1998), colorimetric screening of bacterial colonies or plaques (Kuritz, 1999), in vitro expression cloning (King et al., 1997) and cell surface or phage display (Benhar, 2001). In some cases, the screening method may be a method selected from the following: yeast two-hybrid, n-hybrid, reverse two-hybrid, reverse n-hybrid, split two hybrid, bacterial display, phage display, retroviral display, ribosome display, covalent display, in vitro display or any other display method. In some cases, libraries are screened using phage display methods.

[0255] Analysis of sequences derived from template hopping: The bands corresponding to the template hopping products can be excised from polyacrylamide gels, eluted with sodium acetate (e.g., 0.3 M sodium acetate, pH 5.2), SDS (e.g., 0.03%) at room temperature for several hours, phenol / chloroform extracted, and ethanol precipitated. The isolated cDNA can then be used as a template for PCR amplification using one or more primers. The PCR products can then be cloned directly into a vector (Burke et al., R4: A non-LTR Retrotransposon Specific to the Large Subunit rRNA Gene of Nematodes, Nucleic Acids Res. 23:4628-4634 (1995)), and each clone is sequenced.

[0256] In some cases, variants or modified enzymes, or non-natural enzymes, or modified polypeptides have improved enzyme properties compared to unmodified, wild-type or naturally occurring enzymes or polypeptides. In some cases, the improved enzyme properties are selected from at least one of the following: improved stability (e.g., improved thermostability), improved specific activity, increased protein expression, improved purification, improved processivity, improved strand displacement, improved template hopping, improved ssDNA initiation, and improved fidelity. In some cases, the term stability may include, but is not limited to, thermostability, storage stability, and pH stability. In some cases, specific activity is a measure of the enzymatic activity of a protein or enzyme (in units) relative to the total amount of the protein or enzyme used in the reaction. In some cases, specific activity is measured based on the ability of an enzyme to produce cDNA molecules. In some cases, specific activity is measured in U / mg protein determined based on a primer extension reaction. In some cases, the changed or improved properties can be characterized by a performance index (PI), wherein the PI is a performance ratio of a variant, a modified enzyme, or a non-naturally occurring enzyme compared to a wild-type or compared to a naturally occurring enzyme or protein. The term "performance index (PI)" may refer to the performance ratio of a variant polypeptide to a parent polypeptide, or a modified enzyme to an unmodified enzyme (e.g., a reverse transcriptase), or a non-naturally occurring enzyme to a naturally occurring enzyme for a specified performance characteristic. In some cases, the specified performance or enzyme characteristic characteristics may include, but are not limited to, stability (e.g., thermostability), specific activity, protein expression, purification, processivity, end-to-end template hopping, improved ssDNA priming, and / or fidelity. In some cases, the PI exceeds about 0.5, while in other cases, the PI is about 1 or greater than about 1. In some cases, the variant polypeptide, modified enzyme (e.g., a modified reverse transcriptase), or non-naturally occurring enzyme comprises a modification at one or more amino acid positions. In some cases, the modified enzyme or non-naturally occurring enzyme has a performance index (PI) of about 0.1 or greater, about 0.2 or greater, about 0.3 or greater, about 0.4 or greater, about 0.5 or greater, about 0.6 or greater, about 0.7 or greater, about 0.8 or greater, about 0.9 or greater, about 1 or greater, about 1.2 or greater, about 1.5 or greater, about 2, about 2.5 or greater, about 3 or greater, about 3.5 or greater, about 4, about 4.5 or greater, about 5, about 5.5 or greater, about 6, about 6.5 or greater, about 7, about 8, about 9 or greater, about 10, about 50 or greater, about 75 or greater, about 100 or greater, about 500 or greater, about 1000 or greater.In some cases, the performance index (PI) of the variant or modified enzyme is about 0.1 to about 1, about 0.5 to about 1, about 0.1 to about 2, about 1 to about 2, about 0.5 to about 2, about 0.5 to about 10, about 1 to about 10, about 0.1 to about 10, about 1 to about 5, about 0.5 to about 5, about 0.5 to about 20, about 0.3 to about 20, about 5 to about 10, about 1.5 to about 10, about 1.5 to about 50, about 1 to about 50, about 1.5 to about 100, about 1.5 to about 75, about 4 to about 10, 3 to about 10, about 3 to about 25, about 3 to about 50, about 2 to about 20, about 2 to about 100, about 2 to about 1000, about 1 to about 1000. In some cases, the performance index is determined for protein expression. In some cases, a performance index is determined for the characteristics of at least one improved enzyme property. In some cases, a performance index is determined for purification. In some cases, a performance index is determined for stability (e.g., thermostability). In some cases, a performance index is determined for specific activity. In some cases, a performance index is determined for processivity. In some cases, a performance index is determined for strand displacement. In some cases, a performance index is determined for template jumping. In some cases, a performance index is determined for fidelity. In some cases, the characteristics of the improved enzyme property are selected from the group consisting of: improved thermostability, improved specific activity, and increased protein expression. In some cases, the performance index is determined at 30°C. In some cases, enzyme properties are determined at 30°C. In some cases, enzyme properties, stability (e.g., thermostability), specific activity, protein expression, purification, processivity, strand displacement, template jumping, and / or fidelity are determined at 30°C. In some cases, the performance index for measuring enzyme properties is determined at a specific temperature. In some cases, the temperature is about 25°C to about 42°C. In some cases, the temperature is about 8°C to about 50°C. In some cases, performance indicators for measuring enzyme properties can be determined at temperatures ranging from about 8°C to about 50°C, about 12°C to about 42°C, about 25°C to about 42°C, about 25°C to about 40°C, about 28°C to about 38°C, about 30°C to about 38°C, about 35°C to about 37°C, about 27°C to about 38°C, about 27°C to about 37°C, about 26°C to about 42°C, about 25°C to about 38°C, about 27°C to about 38°C, about 29°C to about 38°C, about 29°C to about 32°C.In some cases, the performance index for measuring enzyme properties can be determined at a temperature of about 8° C. or less, about 12° C. or less, about 20° C. or less, about 4° C. or less, about 55° C. or less, about 37° C. or less, about 25° C. or less, about 28° C. or less, about 30° C. or less, about 32° C. or less, about 34° C. or less, about 35° C. or less, about 36° C. or less, about 33° C. or less, about 31° C. or less, about 60° C. or less, about 38° C. or less, about 39° C. or less, about 40° C. or less, about 41° C. or less, about 42° C. or less, about 50° C. or less. In some cases, the temperature can be in the range of about 25° C. to about 80° C.

[0257] In some cases, the modified enzyme has a specific activity of about 5 units / mg to about 140,000 units / mg, about 5 units / mg to about 125,000 units / mg, about 50 units / mg to about 100,000 units / mg, about 100 units / mg to about 100,000 units / mg, about 250 units / mg to about 100,000 units / mg, about 500 units / mg to about 100,000 units / mg, about 1000 units / mg to about 100,000 units / mg, about 5000 units / mg to about 100,000 units / mg, about 10,000 units / mg to about 100,000 units / mg, about 25,000 units / mg to about 75,000 units / mg. In some cases, the range of specific activity includes about 20,000 units / mg to about 140,000 units / mg, about 20,000 units / mg to about 130,000 units / mg, about 20,000 units / mg to about 120,000 units / mg, about 20,000 units / mg to about 110,000 units / mg, about 20,000 units / mg to about 100,000 units / mg, about 20,000 units / mg to about 90,000 units / mg, or about 100,000 units / mg. The specific activity of the invention can range from about 25,000 Units / mg to about 140,000 Units / mg, from about 25.0 Units / mg to about 130,000 Units / mg, from about 25,000 Units / mg to about 120,000 Units / mg, from about 25,000 Units / mg to about 110,000 Units / mg, from about 25,000 Units / mg to about 100,000 Units / mg, and from about 25,000 Units / mg to about 90,000 Units / mg. In some cases, the lower end of the specific activity range can vary between 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, and 80,000 units / mg. In some cases, the upper end of the range can vary between 150,000, 140,000, 130,000, 120,000, 110,000, 100,000, and 90,000 units / mg.

[0258] Reagent test kit

[0259] Any composition described herein may be included in the kit. In non-limiting examples, the kit in a suitable container comprises one or more primers. The kit may also include reaction components (e.g., dNTPs, polymerases, buffers) for primer extension and amplification. The kit may include reagents (e.g., primers (probes), dNTPs, polymerases, and enzymes) for library formation. The kit may also include methods for purification, such as bead suspensions. The kit may include reagents for sequencing, such as fluorescently labeled dNTPs, sequencing primers, etc.

[0260] In some cases, some components of the test kit can be packaged into aqueous media or lyophilized forms. The container of the test kit can include at least one vial, test tube or other container, in which the component can be placed and appropriately divided. In the case where there is more than one component in the test kit, the test kit can also contain a second, third or other additional container that can be placed separately for other components. When the components of the test kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution. The components of the test kit can be provided in a dry powder form. When reagents and / or components are provided in a dry powder form, the powder can be reconstituted by adding a suitable solvent.

[0261] The kit may include instructions for employing the kit components and for using any additional reagents not included in the kit. The instructions may include variations that may be performed.

[0262] In some cases, the kit can be used to prepare cDNA from a template (e.g., an RNA template). Such a kit can include a carrier device that is separated to accommodate one or more containers, such as vials, test tubes, etc., each container including a separate element for preparing cDNA from RNA. For example, a first container can be provided, and its contents include reverse transcriptase (e.g., non-retroviral reverse transcriptase, non-LTR retrotransposon, R2 reverse transcriptase) or its variants, in liquid solution, powder form, or lyophilized form. In addition, many additional containers can be provided, and their contents independently include a suitable buffer, a substrate for synthesizing nucleotides (e.g., deoxynucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP) individually or together in a suitable solution), a template (e.g., a template RNA), one or more primers and a receptor nucleic acid molecule (e.g., a receptor RNA), and a terminal transferase that is optionally in a solution. In some cases, the kit may include fragments or degraded nucleic acids, DNA, RNA, or combinations thereof, one of a plurality of primers, a receptor nucleic acid molecule (e.g., a receptor nucleic acid molecule comprising modified nucleotides), a reverse transcriptase (e.g., a non-retroviral reverse transcriptase, a non-LTR retrotransposon, an R2 reverse transcriptase) or a variant thereof, a suitable buffer, a substrate for synthesizing nucleotides (e.g., a deoxynucleotide triphosphate (e.g., dATP, dCTP, dGTP, and dTTP)). Any combination of the above components may be provided. The kit may not include any of the above components. In some cases, one or more primers may be one or more random primers. In some cases, any component may be individually loaded.

[0263] The present disclosure relates to a kit for producing nucleic acid molecules (e.g., cDNA molecules), comprising: one or more primers, nucleotides, at least one modified reverse transcriptase, a template, and instructions for performing any of the methods disclosed in the present disclosure. In some cases, the kit can be used to detect nucleic acids, the kit comprising a nucleic acid template (e.g., a DNA template), at least one modified reverse transcriptase, nucleotides, and instructions for performing any of the methods disclosed in the present disclosure. In some cases, the modified reverse transcriptase present in or used in the kit is active and / or capable of template hopping at a temperature equal to or less than about, or greater than about 4°C, 8°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 52°C, 55°C, or 60°C. In some cases, nucleic acid and / or template (such as nucleic acid template, DNA or RNA) exist with following concentration: as low as about 50 femtomoles, as low as about 60 femtomoles, as low as about 70 femtomoles, as low as about 75 femtomoles, as low as about 80 femtomoles, as low as about 90 femtomoles, as low as about 100 femtomoles, as low as about 120 femtomoles, as low as about 150 femtomoles, as low as about 200 femtomoles, as low as about 250 femtomoles, as low as about 300 femtomoles, as low as about 350 femtomoles, as low as about 400 femtomoles, as low as about 500 femtomoles, as low as about 550 femtomoles, as low as about 600 femtomoles, as low as about 700 femtomoles or as low as about 800 femtomoles. In some cases, nucleic acid and / or template (such as nucleic acid template, DNA or RNA) exist with concentration up to 1 micromole. In some cases, test kit can include one or more primers and / or anneal to the template of primer. The disclosure also relates to a test kit for producing modified enzyme, modified reverse transcriptase or modified polypeptide. In some cases, the kit includes a PCR step and / or components for PCR.

[0264] In some cases, the disclosure relates to a kit for detecting nucleic acids, the kit comprising a template, at least one modified reverse transcriptase, nucleotides, and instructions for performing the methods of the disclosure. In some cases, the nucleic acid is present at a concentration of at least about 50 femtomolar, at least about 20 femtomolar, at least about 100 femtomolar, or greater than about 1000 femtomolar.

[0265] Sequencing

[0266] In some cases, determining the number of different labeled nucleic acids can include determining the sequence of the labeled nucleic acid or any product thereof (e.g., labeled amplicon, labeled cDNA molecule). In some cases, the amplified target nucleic acid can be sequenced. Determining the sequence of the labeled nucleic acid or any product thereof can include performing a sequencing reaction to determine the sequence of at least a portion of the sample tag, the molecular identification tag, at least a portion of the labeled nucleic acid, its complement, its reverse complement, or any combination thereof. In some cases, only the sample tag or a portion of the sample tag is sequenced. In some cases, only the molecular identification tag or a portion of the molecular identification tag is sequenced.

[0267] The sequence of a labeled nucleic acid or any product thereof can be determined by sequencing methods such as Helioscope (registered trademark) single-molecule sequencing, nanopore DNA sequencing, massively parallel signature sequencing (MPSS) by Lynx Therapeutics, 454 pyrosequencing, single-molecule real-time (RNAP) sequencing, Illumina (Solexa) sequencing, SOLiD sequencing, IonTorrent, ion semiconductor sequencing, single-molecule SMRT (registered trademark) sequencing, Polony sequencing, DNA nanoball sequencing, and methods by VisiGen Biotech. Alternatively, the sequence of the labeled nucleic acid or any product thereof can be determined using a sequencing platform, including but not limited to Genome Analyzer IIx, HiSeq and MiSeq provided by Illumina; single molecule real-time (SMRT (registered trademark)) technology, such as the PacBio RS system provided by Pacific Biosciences (California), and Solexa sequencer; accurate single molecule sequencing (True Single Molecule Sequencing; tSMS (registered trademark)) technology, such as the HeliScope (registered trademark) sequencer provided by Helicos (Cambridge, Mass.). In some cases, sequencing reactions can occur on solid or semi-solid supports, in gels, in emulsions, on surfaces, on beads, in droplets, in continuous tracking, in diluents, or in one or more physically separated volumes.

[0268] Sequencing can comprise sequencing at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides or base pairs of the labeled nucleic acid. In some cases, sequencing comprises sequencing at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides or base pairs of the labeled nucleic acid. In other cases, sequencing comprises sequencing at least about 1500, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 or more nucleotides or base pairs of the labeled nucleic acid.

[0269] The sequencing may comprise at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more sequencing reads per run. In some cases, the sequencing comprises sequencing at least about 1500, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 or more sequencing reads per run. The sequencing may comprise less than or equal to about 1,600,000,000 sequencing reads per run. The sequencing may comprise less than or equal to about 200,000,000 reads per run.

[0270] cell

[0271] Cells as described in the present disclosure can be cells from animals (e.g., humans, rats, pigs, horses, cows, dogs, mice). In some cases, cells can be single cells. In some cases, cells are human cells. Cells can be human fetal cells. Human fetal cells can be obtained from a mother with a fetus. Cells can be cells from a pregnant mother. Cells can be cells from vertebrates, invertebrates, fungi, archaea, or bacteria. Cells can be from multicellular tissues (e.g., organs (e.g., brain, liver, lungs, kidneys, prostate, ovaries, spleen, lymph nodes, thyroid, pancreas, heart, skeletal muscle, intestines, larynx, esophagus, and stomach), blastocysts). Cells can be cells from cell cultures. Cells can be HeLa cells, K562 cells, Ramos cells, hybridomas, stem cells, undifferentiated cells, differentiated cells, circulating cells, CHO cells, 3T3 cells, and the like.

[0272] Circulating pathological cells that can be used for the disclosed method include all types of circulating cells that may be affected by a disease or condition or infected by an infectious agent. Circulating cells refer to cells present in body fluids. Circulating cells may not necessarily circulate throughout the body or circulatory system. For example, circulating cells may be present in local areas, such as synovial fluid or cerebrospinal fluid or lymph fluid. Circulating pathological cells can also be separated from tissues or organs that have been affected by a disease or condition or infected by an infectious agent. In other cases, circulating pathological cells can be a mixture of different types of circulating pathological cells.

[0273] In some cases, the cell is a cancer cell. Non-limiting examples of cancer cells can include prostate cancer cells, breast cancer cells, colon cancer cells, lung cancer cells, brain cancer cells and ovarian cancer cells. In some cases, the cell is from cancer (e.g., circulating tumor cells). Non-limiting examples of cancer can include adenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, chondrosarcoma and fibrosarcoma.

[0274] In some cases, the cell is a rare cell. Rare cells can be circulating tumor cells (CTCs), circulating epithelial cells (CECs), circulating stem cells (CSCs), stem cells, undifferentiated stem cells, cancer stem cells, bone marrow cells, progenitor cells, foam cells, fetal cells, mesenchymal cells, circulating endothelial cells, circulating endometrial cells, trophoblasts, immune system cells (host or transplant cells), connective tissue cells, bacteria, fungi or pathogens (e.g., bacteria or protozoa), microparticles, cell fragments, proteins and nucleic acids, organelles, other cellular components (e.g., mitochondria and nuclei) and viruses.

[0275] In some cases, the cell is from a tumor. In some cases, the tumor is benign or malignant. Tumor cells may include metastatic cells. In some cases, the cell is from a solid tissue comprising a plurality of different cell types (e.g., different genotypes).

[0276] sample

[0277] In some cases, samples including template nucleic acid (such as DNA and / or RNA) may be merged into reaction mixtures in an amount sufficient to produce product. In some cases, samples are merged into reaction mixtures so that the ultimate concentration of DNA and / or RNA in the reaction mixture is about 1 fg / μL to about 10 μg / μL, about 1 μg / μL to about 5 μg / μL, about 0.001 μg / μL to about 2.5 μg / μL, about 0.005 μg / μL to about 1 μg / μL, about 0.01 μg / μL to about 0.5 μg / μL, about 0.1 μg / μL to about 0.25 μg / μL. In some cases, samples including templates are separated from single cells. In some cases, samples including templates are separated from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500 or more cells.

[0278] In some cases, the template is a combination of DNA, RNA, or DNA and RNA. In some cases, the template is a fragment or degraded DNA, a fragment or degraded RNA, or a combination of a fragment or degraded DNA and a fragment or degraded RNA. In some cases, the total amount of template is the total amount of template in a sample. In some cases, the total amount of template is the total amount of template in a reaction mixture. In some cases, the total amount of template is the total amount of template in a pot or a single container. In some cases, the total amount of template is the total amount of template in a pot or a single container reaction. In some cases, the total amount of template is about 1 femtomolar (fM) to about 100 micromolar, about 0.0001 micromolar to about 0.01 micromolar, about 0.0001 micromolar to about 0.1 micromolar, about 40 femtomolar to about 0.01 micromolar, about 50 femtomolar to about 500 femtomolar, about 50 femtomolar to about 0.01 micromolar, about 50 femtomolar to about 0.1 micromolar, about 50 femtomolar to about 500 picomolar, about 50 femtomolar to about 500 nanomolar, about 50 femtomolar to about 500 micromolar, about 50 femtomolar to about 1 picomolar, about 40 femtomolar to about 1 nanomolar, about 1 femtomolar to about 1 picomolar.In some cases, the total amount of template is equal to or is at least about or less than about 1000 micromolar, equal to or is at least about or less than about 500 micromolar, equal to or is at least about or less than about 250 micromolar, equal to or is at least about or less than about 100 micromolar, equal to or is at least about or less than about 50 micromolar, equal to or is at least about or less than about 25 micromolar, equal to or is at least about or less than about 10 micromolar, equal to or is at least about or less than about 1 micromolar, equal to or is at least about or less than about 0.1 micromolar, equal to or is at least about or less than about 0.01 micromolar, equal to or is at least about or less than about 0.001 micromolar, equal to or is at least about or less than about less than about 0.0001 micromolar, equal to or at least about or less than about 2000 nanomolar, equal to or at least about or less than about 500 nanomolar, equal to or at least about or less than about 250 nanomolar, equal to or at least about or less than about 200 nanomolar, equal to or at least about or less than about 50 nanomolar, equal to or at least about or less than about 25 nanomolar, equal to or at least about or less than about 20 nanomolar, equal to or at least about or less than about 2 nanomolar, equal to or at least about or less than about 0.2 nanomolar, equal to or at least about or less than about 0.01 nanomolar, equal to or at least about or less than about 0.001 nanomolar, equal to or at least about about or less than about 0.0001 nanomoles, equal to or at least about or less than about 3000 picomoles, equal to or at least about or less than about 500 picomoles, equal to or at least about or less than about 250 picomoles, equal to or at least about or less than about 300 picomoles, equal to or at least about or less than about 50 picomoles, equal to or at least about or less than about 25 picomoles, equal to or at least about or less than about 30 picomoles, equal to or at least about or less than about 3 picomoles, equal to or at least about or less than about 0.3 picomoles, equal to or at least about or less than about 0.01 picomoles, equal to or at least about or less than about 0.001 picomoles, equal to or At least about or less than about 0.0001 picomoles, equal to or at least about or less than about 5000 femtomoles, equal to or at least about or less than about 500 femtomoles, equal to or at least about or less than about 250 femtomoles, equal to or at least about or less than about 50 femtomoles, equal to or at least about or less than about 25 femtomoles, equal to or at least about or less than about 10 femtomoles, equal to or at least about or less than about 1 femtomoles, equal to or at least about or less than about 0.1 femtomoles, equal to or at least about or less than about 0.01 femtomoles, equal to or at least about or less than about 0.001 femtomoles, equal to or at least about or less than about 0.0001 femtomoles.

[0279] In some cases, the sample can be obtained from a biological sample obtained from a subject. In some cases, the sample comprises a circulating tumor DNA sample and / or a tissue sample. In some cases, the biological sample comprises a free biological sample. In some cases, the biological sample comprises a circulating tumor DNA sample. In some cases, the biological sample comprises a biopsy sample. In some cases, the biological sample comprises a tissue sample. In some cases, the biological sample comprises a liquid biopsy. In some cases, the biological sample comprises free DNA. In some cases, the biological sample can be a solid biological sample, such as a tumor sample. In some cases, a sample from a subject may contain at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% tumor cells or nucleic acids from a tumor. A solid biological sample can be processed by fixing in a formalin solution and then embedding in paraffin (e.g., a FFPE sample). A solid biological sample can be processed by freezing. Alternatively, a biological sample can be neither fixed nor frozen. An unfixed, unfrozen sample can be stored in a solution configured to preserve nucleic acids. The solid biological sample may optionally be homogenized, sonicated, French pressed, dounced, frozen / thawed, and may then be centrifuged.

[0280] In some cases, the sample can be a liquid biological sample. In some cases, the liquid biological sample can be a blood sample (e.g., whole blood, plasma, or serum). The whole blood sample can be separated from the cellular components (e.g., plasma, serum) and cellular components by using Ficoll reagent. In some cases, the liquid biological sample can be a urine sample. In some cases, the liquid biological sample can be a perilymph sample. In some cases, the liquid biological sample can be a fecal sample. In some cases, the liquid biological sample can be saliva. In some cases, the liquid biological sample can be semen. In some cases, the liquid biological sample can be amniotic fluid. In some cases, the liquid biological sample can be cerebrospinal fluid. In some cases, the liquid biological sample can be bile. In some cases, the liquid biological sample can be sweat. In some cases, the liquid biological sample can be tears. In some cases, the liquid biological sample can be sputum. In some cases, the liquid biological sample can be synovial fluid. In some cases, the liquid biological sample can be vomit. In some cases, the liquid biological sample can be a free sample. In some specific cases, the free sample can be a free plasma sample.

[0281] The polynucleotides in the sample (which may be referred to as input nucleic acids or inputs) may include DNA. The input nucleic acids may be composite DNA, such as double-stranded DNA, genomic DNA, or mixed nucleic acids from more than one organism. The polynucleotides in the sample may include RNA. RNA may be obtained and purified. RNA may include RNA in purified or unpurified form, including but not limited to mRNA, tRNA, snRNA, rRNA, retrovirus, small non-coding RNA, microRNA, polyribosomal RNA, mRNA precursor, intron RNA, viral RNA, free RNA, and fragments thereof. Non-coding RNA or ncRNA may include snoRNA, microRNA, siRNA, piRNA, and long ncRNA. The polynucleotides in the sample may include cDNA. cDNA may be generated from RNA (e.g., mRNA). cDNA may be single-stranded or double-stranded. The input DNA may be mitochondrial DNA. The input DNA may be free DNA. Cell-free DNA may be obtained from, for example, serum or plasma samples. The input DNA may be from more than one individual or organism. The input DNA may be double-stranded or single-stranded.

[0282] In some cases, samples can be collected over a period of time. Samples can be collected at regular time intervals or can be collected intermittently at irregular time intervals. Nucleic acids from different samples can be compared, for example, to monitor the progression or recurrence of a condition or disease.

[0283] In some cases, the sample can be collected by core biopsy. In some cases, the sample can be collected in the form of purified nucleic acid. Examples of these purified samples can include precipitated nucleic acid attached to filter paper, phenol-chloroform extraction, nucleic acid purified by kit purification (e.g., Quigen Miniprep (registered trademark) etc.) or gel-purified nucleic acid as illustrative examples.

[0284] The sample of the present disclosure can be a sample from an animal (e.g., human, rat, pig, horse, cow, dog, mouse). In some cases, the sample is a human sample. The sample can be a human fetal sample. The sample can be from a multicellular tissue (e.g., an organ (e.g., brain, liver, lung, kidney, prostate, ovary, spleen, lymph node, thyroid, pancreas, heart, skeletal muscle, intestine, larynx, esophagus, and stomach), blastocyst). The sample can be a cell from a cell culture.

[0285] A sample may contain multiple cells. A sample may contain multiple cells of the same type. A sample may contain multiple cells of different types. A sample may contain multiple cells at the same point in the cell cycle and / or differentiation pathway. A sample may contain multiple cells at different points in the cell cycle and / or differentiation pathway. A sample may contain multiple samples.

[0286] The plurality of samples may contain one or more malignant cells. The one or more malignant cells may be derived from a tumor, a sarcoma, or a leukemia.

[0287] The plurality of samples may include at least one body fluid. The body fluid may include blood, urine, lymph, saliva. The plurality of samples may include at least one blood sample.

[0288] The plurality of samples may comprise at least one cell from one or more biological tissues. The one or more biological tissues may be bone, heart, thymus, artery, blood vessel, lung, muscle, stomach, intestine, liver, pancreas, spleen, kidney, gallbladder, thyroid, adrenal gland, breast, ovary, prostate, testis, skin, fat, eye, or brain.

[0289] The biological tissue may include infected tissue, diseased tissue, malignant tissue, calcified tissue or healthy tissue.

[0290] In some cases, the feature of improved enzyme properties is selected from the group consisting of increased stability (e.g., increased thermostability), increased specific activity, increased protein expression, increased processivity, increased strand displacement, increased end-to-end template hopping, and increased fidelity.

[0291] Examples

[0292] The following specific examples are illustrative and non-limiting. The examples described herein refer to the various embodiments described in the preceding sections and provide non-limiting support for these embodiments.

[0293] Example 1 : Expression and purification

[0294] Small and medium scale: The expression vector pET-45b for one of the modified R2 reverse transcriptases of the modified R2 non-long terminal repeat (LTR) retrotransposon or SEQ ID No: 1-20 is transformed into Escherichia coli BL21 (DE3). Table 1 below shows examples of non-naturally occurring R2 enzyme variants of the present disclosure. For expression, the preculture is set in 2ml LB containing 100μM carbenicillin and grown overnight at room temperature for about 8 to 12 hours. After about 8 to 12 hours, 200μL of the preculture can be transferred to 25mL of the self-inducing expression medium Overnight ExpressTB (Novagen) and cultured at room temperature for 36 to 48 hours with shaking. The cells are harvested by centrifugation at 8000×g for 10 minutes at 4-8°C. The biomass centrifuge block is frozen for a minimum of 1 hour at -20°C.

[0295] Purification: The pellet was resuspended in 0.5 mL of lysis buffer (0.5 mL of lysis buffer per 1 / 6 biomass) and incubated at room temperature for 30 minutes. Lysis buffer composition: 1x BugBuster, 100 mM sodium phosphate, 0.1% Tween, 2.5 mM TCEP, 3 μL protease inhibitor cocktail (Roche), 50 μg lysozyme, 0.5 μL DNaseI (2,000 units / ml from NEB). After incubation, the lysate can be mixed with an equal volume (0.5 mL) of His binding buffer (50 mM sodium phosphate pH 7.7, 1.5 M sodium chloride, 2.5 mM TCEP, 0.1% Tween, 0.03% Triton X-100 and 10 mM imidazole) and incubated at room temperature for about 10-15 minutes. After incubation, the lysate can be centrifuged at 10,000×g for about 15 minutes at a temperature of about 4° C. to about 8° C. The pellet can then be mixed with 250 μL of His affinity gel (His-Spin Protein Miniprep from Zymo Research) according to the manufacturer's protocol. After the binding step, the His affinity gel is washed three times with wash buffer (50 mM sodium phosphate pH 7.7, 750 mM sodium chloride, 0.1% Tween, 0.03% Triton X-100, 2.5 mM TCEP, and 50 mM imidazole). R2 reverse transcriptase (RT) (e.g., a non-naturally occurring enzyme) can be eluted with 150 μL of elution buffer (50 mM sodium phosphate pH 7.7, 300 mM sodium chloride, 2.5 mM TCEP, 0.1% Tween, and 250 mM imidazole) and used directly or frozen in 30% glycerol. This protocol can be adapted for mutagenic expression and purification and for screening. For example, a similar protocol can be adapted to a plate format, e.g., 2 mL Overnight Express TB (Novagen) can be used instead of 25 mL, and the purification step can involve a 96-well spin plate containing nickel-immobilized resin.

[0296] Results: After purification, samples were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 4-12% polyacrylamide, Bis-Tris.

[0297] Example 2 : Surrogate / diagnostic assays

[0298] Example of reverse transcriptase (RT) activity assay: The activity assay can be used to compare the enzyme activity, activity fraction, stability (e.g., thermostability), and robustness of non-naturally occurring enzymes. RT activity and activity fraction can be estimated based on a primer extension assay by comparing the fraction of DNA primer extended to that not extended using various template / primer and enzyme concentrations. The extension assay is performed with and without the addition of a DNA trap.

[0299] Example protocol: 0.2 μM template / primer annealed with a fluorescently labeled primer can be pre-incubated with various concentrations of R2 RT (0.1 to 4 times relative to template / primer) for 20 minutes at room temperature. Pre-incubation conditions can include 40 mM Tris pH 7.5, 200 mM NaCl, 5 mM TCEP and 0.1% Tween. Extension can begin with the addition of MgCl2 (5 mM, final) and dNTPs (25 μM each, final) and optional DNA capture agent (final 3 μM unlabeled DNA oligonucleotide duplex or heparin). Adding a DNA capture agent helps estimate the RT activity score. The reaction is then incubated for 10 minutes and stopped with EDTA (50 mM, final) or formamide (50%, final). The reaction products can then be analyzed using 15% PAGE-urea. An example of a template sequence used is rCrArG rUrCrA rGrUrC rArGrU rCrArG rUrCrA rGrUrG rCrCrA rArArUrGrCrC rUrCrG rUrCrA rUrC, and an example of a primer is / 56-FAM / TGATGACGAGGCATTTGGC.

[0300] Example of an end-to-end template hopping assay: A primer extension assay is performed using two templates, one of which is annealed to a fluorescently labeled primer (the donor template) and the other template contains no primer (the acceptor nucleic acid).

[0301] Example protocol: 0.1 μM template / primer annealed with a fluorescently labeled primer (alternatively, the reaction product can be stained with Syber Gold) is pre-incubated with various concentrations of R2 RT (0.1 to 4 times relative to the template / primer) at room temperature for 20 minutes. Pre-incubation conditions can include 40 mM Tris pH 7.5, 200 mM NaCl, 5 mM TCEP, and 0.1% Tween. Extension can begin with the addition of MgCl2 (5 mM, final), dNTPs (50 μM each, final), and various concentrations (ranging from about 0.01 μM to about 5 μM) of the receptor nucleic acid. The reaction is then incubated for 30 minutes to 1 hour and stopped with EDTA (50 mM, final) or formamide (50%, final). The reaction products can be analyzed using 15% PAGE-urea.

[0302] template : T7 RNA polymerase can be used to generate templates by in vitro RNA synthesis based on DNA templates generated by two primers in a PCR reaction, one of which includes a T7 promoter sequence (i.e., the first primer). The second primer can also be used as a DNA primer in a donor template / primer scheme. The reaction product can then be analyzed using 15% PAGE-urea. Examples of materials used: template pUC18 for PCR amplification using T7 primer CTGCAGTAATACGACTCACTATAGGATCCTCTAGAGTCGACCTGC (SEQ ID NO: 24); donor primer GCCATTCGCCATTCAGGCTGC (SEQ ID NO: 102) (used for PCR amplification and priming at donor RNA template); RNA template (about 190 nucleotides); acceptor nucleic acid-G blocking PCR template ACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGTACCGCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCC (SEQ ID NO: 103); two primers for PCR amplification (T7 primer ACGGCCAGTGAATTGTAATACGAC (SEQ ID NO: 104) and a second primer GGAAACAGCTATGACCATG (SEQ ID NO: 105)).

[0303] Examples of processivity analysis: The processivity of each non-naturally occurring enzyme can be analyzed based on primer extension and product formation using 15% PAGE-urea or 1.2% agarose gel or 2% agarose gel. Product length distribution can be analyzed using densitometry.

[0304] Example Protocol: 0.05-0.1 μM template / primer annealed with a fluorescently labeled primer (alternatively, the reaction product can be stained with Syber Gold) can be pre-incubated with various concentrations of R2 RT (0.1 to 4 times relative to template / primer) for 20 minutes at room temperature. Pre-incubation conditions: 40 mM Tris pH 7.5, 200 mM NaCl, 5 mM TCEP, and 0.1% Tween. Extension can be initiated with the addition of MgCl2 (5 mM, final), dNTPs (50 μM each, final), and optional DNA capture agent (3 μM final unlabeled DNA oligonucleotide duplex). The reaction can then be incubated for 30 minutes to 1 hour and stopped with EDTA (50 mM, final) or formamide (50%, final).

[0305] Template: Template can be generated by in vitro RNA synthesis using T7 RNA polymerase based on DNA template generated using two primers in PCR reaction, one of which includes T7 promoter sequence. The second primer can also be used as DNA primer in donor template / primer scheme. The reaction product is analyzed using 15% PAGE-urea or 1.2% agarose gel or 2% agarose gel. Materials include: template pUC18 for PCR amplification using T7 primer CTGCAGTAATACGACTCACTATAGGATCCTCTAGAGTCGACCTGC, RT primer CAGGGTTATTGTCTCATGAGCG (SEQ ID NO: 101) (for PCR amplification and priming at donor RNA template); and RNA template (about 600 nucleotides).

[0306] Examples of random priming: long RNA template with several primers containing adapters or random primers containing adapters; PCR amplification followed by product analysis to compare product length distribution (one primer is specific to the 5' end of the template and...

Claims

1. A non-naturally occurring enzyme comprising (i) a finger domain derived from an R2 retrotransposon; (ii) a thumb domain derived from an R2 retrotransposon; and (iii) an endonuclease domain derived from an R2 retrotransposon.

2. The non-naturally occurring enzyme of claim 20, wherein the non-naturally occurring enzyme has at least 80% identity to SEQ ID NO: 1-20.

3. A method for simultaneously amplifying ribonucleic acid (RNA) molecules and deoxyribonucleic acid (DNA) molecules, comprising: (a) providing a reaction mixture comprising the RNA, DNA, and a non-naturally occurring enzyme, each of the non-naturally occurring enzymes comprising (i) a finger domain derived from a non-retroviral transposon or from an R2 retrotransposon; (ii) a thumb domain derived from an R2 retrotransposon; (iii) a palm domain derived from an R2 retrotransposon; and (iv) an endonuclease domain derived from an R2 retrotransposon; and (b) subjecting the reaction mixture to conditions sufficient to amplify the RNA and the DNA, thereby obtaining amplification products of the RNA and the DNA.

4. The method of claim 3, wherein the DNA is complementary DNA derived from a subset of RNA in the reaction mixture.

5. A method for preparing a complementary deoxyribonucleic acid (cDNA) molecule, comprising: (a) partitioning a cell and a non-naturally occurring reverse transcriptase in the partitions, wherein the cell comprises a ribonucleic acid (RNA) molecule; (b) releasing the RNA molecules from the cells in the partitions; and (c) in the partition, synthesizing a complementary deoxyribonucleic acid (cDNA) library from the RNA molecule using the non-naturally occurring reverse transcriptase, wherein the non-naturally occurring transcriptase synthesizes the cDNA library with a processivity of 20 nucleotides or longer.

6. The method of claim 3, wherein the non-naturally occurring reverse transcriptase has at least 80% identity to SEQ ID NOs: 1-20.

7. The method of claim 3, wherein the partitioning further comprises: i. one or more receptor nucleic acid molecules; and ii. A non-naturally occurring reverse transcriptase, wherein the non-naturally occurring reverse transcriptase has at least 80% identity to SEQ ID NO: 1-20.

8. A method for processing a sample containing different types of ribonucleic acids (RNA), comprising synthesizing complementary deoxyribonucleic acid (cDNA) molecules using said RNA molecules in the presence of ribosomal ribonucleic acid (rRNA) molecules that are blocked for transcription, such that less than 30% of said cDNA molecules contain sequences from said rRNA molecules.

9. A method for treating a mixture containing ribonucleic acid (RNA), comprising: (a) fragmenting the RNA molecule in the mixture to obtain a plurality of RNA fragments; (b) contacting one or more single-stranded nucleic acid sequences with the plurality of RNA fragments, the one or more single-stranded nucleic acid sequences being complementary to at least a subset of the RNA fragments, thereby providing one or more RNA fragment complexes comprising the one or more single-stranded nucleic acid sequences hybridized to said at least said subset of the RNA fragments; and (c) synthesizing at least one complementary deoxyribonucleic acid (cDNA) molecule from the RNA using a reverse transcriptase in the presence of the one or more RNA fragment complexes.

10. A method for sequencing a nucleic acid molecule, comprising: (a) providing a reaction mixture comprising the single-stranded nucleic acid molecule and a non-naturally occurring enzyme, wherein the non-naturally occurring enzyme comprises: i) a finger domain derived from a non-retroviral transposon or from an R2 retrotransposon; ii) the thumb domain derived from the R2 retrotransposon; iii) the palm domain derived from the R2 retrotransposon; and iv) an endonuclease domain derived from the R2 retrotransposon; (b) subjecting the reaction mixture to conditions sufficient to incorporate individual nucleotides into a growing chain complementary to the nucleic acid molecule using the non-naturally occurring enzyme, wherein incorporation of the individual nucleotides into the growing chain results in a detectable signal; and (c) detecting the detectable signal, thereby sequencing the single-stranded nucleic acid molecule. The method of claim 10 , wherein the nucleic acid molecule is an RNA molecule. The method according to claim 10 , wherein the nucleic acid molecule is a single-stranded DNA molecule.

13. The method of claim 10, wherein the conditions sufficient to directly sequence the nucleic acid molecule comprise optics-based single molecule sequencing conditions.

14. The method of claim 10, wherein the conditions sufficient to directly sequence the nucleic acid molecule comprise microscopy-based single molecule sequencing conditions.

15. The method of claim 10, wherein the conditions sufficient to directly sequence the nucleic acid molecule comprise nanopore-based single molecule sequencing conditions.

16. The method of claim 10, wherein the conditions sufficient to directly sequence the nucleic acid molecule comprise field effect transistor-based single molecule sequencing conditions.

17. A method comprising: (a) preparing a complementary deoxyribonucleic acid (cDNA) molecule from one or more ribonucleic acids (RNA) using a naturally occurring enzyme, wherein the one or more ribonucleic acids (RNA) are derived from an in situ tissue of a subject or from a fixed ex vivo tissue of the subject, wherein the non-naturally occurring enzyme comprises: i) a finger domain derived from a non-retroviral transposon or from an R2 retrotransposon; ii) the thumb domain derived from the R2 retrotransposon; iii) the palm domain derived from the R2 retrotransposon; and iv) an endonuclease domain derived from an R2 retrotransposon; thereby producing cDNA molecules from said tissue in situ of said subject or from said fixed ex vivo tissue of said subject; and (b) sequencing the cDNA molecules generated in (a).

18. The method of claim 17, wherein the fixed ex vivo tissue of the subject is fixed in formaldehyde.

19. The method of claim 17, wherein the fixed ex vivo tissue of the subject is fixed in paraffin.

20. A method for preparing a complementary deoxyribonucleic acid molecule, comprising: (a) fragmenting the RNA molecule to obtain multiple RNA fragments; (b) removing a 3'-phosphate group, a 2'-phosphate group and a cyclic 2'3' phosphate from one or more of the RNA fragments, thereby producing one or more dephosphorylated fragmented RNAs; (c) adding a poly-A tail to the one or more dephosphorylated fragmented RNAs; (d) adding the following to the one or more dephosphorylated fragmented RNAs: i. a primer adapter comprising an oligo-T sequence; ii. an acceptor linker; and iii. a non-naturally occurring R2 enzyme having processivity of 20 nucleotides or longer, wherein the non-naturally occurring R2 enzyme reverse transcribes a sequence from the one or more dephosphorylated fragmented RNAs in a 3' to 5' order, wherein upon reaching the 5' end of the one or more dephosphorylated fragmented RNAs the R2 enzyme jumps to the 3'-end of the acceptor adaptor.

21. The method of claim 20, wherein the acceptor linker comprises a nucleotide analog.

22. The method of claim 21, wherein the nucleotide analog is at the 5' end of the acceptor linker.

23. The method of claim 21, wherein the nucleotide analog is at the 5' end of the primer adapter.

24. The method of claim 21, further comprising removing one or more unannealed primer adapters of (d)(i) prior to adding the non-naturally occurring R2 enzyme.

25. The method of claim 21, wherein the one or more unannealed primer adapters are removed using an immobilized poly A oligonucleotide.

26. The method of claim 20, wherein the acceptor linker comprises a 3'-dideoxynucleotide at the 3'-end of the acceptor linker.

27. A method for preparing a complementary deoxyribonucleic acid molecule, comprising: (a) fragmenting a ribonucleic acid (RNA) molecule to obtain a plurality of fragmented RNA fragments; (d) adding the following to the one or more fragmented RNAs: i. Primer adapter; ii. an acceptor linker; and iii. a non-naturally occurring R2 enzyme having processivity of 20 nucleotides or longer, The non-naturally occurring R2 enzyme primes reverse transcription using a plurality of ssDNA primers that are not complementary to the template, wherein upon reaching the 5' end of the fragmented RNA, the R2 enzyme jumps to the 3'-end of the acceptor adaptor.

28. A method for preparing a complementary deoxyribonucleic acid molecule, comprising: (a) Adding the following to unfragmented RNA molecules: i. Primer adapter; ii. an acceptor linker; and iii. a non-naturally occurring R2 enzyme having processivity of 20 nucleotides or longer, The non-naturally occurring R2 enzyme primes reverse transcription using a plurality of ssDNA primers that are not complementary to the template, wherein upon reaching the 5' end of the fragmented RNA, the R2 enzyme jumps to the 3'-end of the acceptor adaptor.

29. A method for depleting a plurality of ribonucleic acids from a sample, comprising: (a) synthesizing complementary deoxyribonucleic acid (cDNA) molecules from RNA templates from the sample, (b) incorporating a first adapter molecule into the 3' end of the synthetic cDNA molecule and incorporating a second adapter molecule into the 5' end of the synthetic cDNA; (c) performing up to 10 cycles of polymerase chain reaction (PCR) using a modified oligonucleotide probe complementary to the rDNA sequence, wherein the modified oligonucleotide probe is configured to allow the probe to bind to a solid support, thereby generating a hybridization product bound to the solid support in the reaction mixture; (d) removing the synthesized cDNA from the reaction mixture while the hybridization product is bound to the solid support, thereby depleting the plurality of ribonucleic acids (RNA) from the sample.

30. The method of claim 29, wherein at most 5 cycles of polymerase chain reaction (PCR) are performed in step (c).

31. The method of claim 30, wherein a single cycle of polymerase chain reaction (PCR) is performed in step (c).

32. The method of claim 29, wherein the plurality of RNAs comprises 5S RNA, 5.8S RNA, 18S RNA, 28S RNA, or 12S mitochondrial RNA.

33. The method of claim 32, wherein the plurality of RNAs comprises at least 2, at least 3, at least 4, or at least 5 of 5S RNA, 5.8S RNA, 18S RNA, 28S RNA, or 12S mitochondrial RNA.

34. The method of claim 29, wherein the modified oligonucleotide probe is a biotin oligonucleotide probe.

35. The method of claim 29, wherein the sample comprises a plurality of fragmented ribonucleic acids (RNA).

36. The method of claim 29, wherein the sample comprises a plurality of full-length ribonucleic acids (RNA).

37. The method of claim 29, further comprising, before step (c), performing an amplification reaction on the cDNA molecules produced in (b).

38. The method of claim 37, wherein the amplification reaction is performed on the cDNA molecules produced in step (b) for up to 10 cycles prior to step (c).

39. The method of claim 29, wherein the cDNA molecule is synthesized using a non-naturally occurring R2 enzyme having a processivity of 20 nucleotides or longer.

40. The method of claim 29, wherein step (c) is performed as an isothermal reaction.

41. A method for depleting a plurality of ribonucleic acids (RNA) from a sample, comprising: (a) synthesizing an asymmetric double-stranded deoxyribonucleic acid molecule that is protected from enzymatic degradation at a first 5' end and is unprotected at a second 5' end from a ribonucleic acid (RNA) molecule by adding the following to a reaction vessel containing the RNA molecule: i. A primer, wherein the primer comprises a modification at its 5' end configured to prevent enzymatic degradation by a 5' to 3' exonuclease; and ii. Enzymes; The synthesis is carried out under conditions sufficient to allow synthesis of the asymmetric double-stranded deoxyribonucleic acid molecule; (b) adding a 5' to 3' exonuclease to the product of step (a), thereby generating ssDNA having a predetermined polarity; and (c) depleting the plurality of RNAs from the product of (b) by hybridizing one of a plurality of probes to the plurality of RNAs and performing a pull-down reaction.

42. The method of claim 41, wherein the plurality of RNAs comprises 5S RNA, 5.8S RNA, 18S RNA, 28S RNA, or 12S mitochondrial RNA.

43. The method of claim 41, wherein the plurality of RNAs comprises at least 2, at least 3, at least 4, or at least 5 of 5S RNA, 5.8S RNA, 18S RNA, 28S RNA, or 12S mitochondrial RNA.

44. The method of claim 41, wherein the sample comprises a plurality of fragmented ribonucleic acids (RNA).

45. The method of claim 41, wherein the sample comprises a plurality of full-length ribonucleic acids (RNA).

46. ​​A method for preparing a sample for RNA sequencing, comprising: (a) Individually labeling multiple single cells with multiple unique barcodes; (b) combining the plurality of single cells in a single pot; (c) performing an RNA sequencing reaction on the plurality of single cells; (d) selecting a cell of interest based on the RNA sequencing reaction and identifying a unique barcode associated with the cell of interest, wherein the selecting and the identifying are performed in a computer program product; (e) hybridizing primers to the unique barcode associated with the cell of interest and performing an amplification reaction in the hybridization sample, thereby generating a plurality of amplicons.

47. The method of claim 46, further comprising selecting a population of cells of interest and identifying the unique barcode associated with each cell from the population.

48. A method for preparing a sample for RNA sequencing, comprising: (a) dissociating a tissue sample in a lysis reaction, thereby generating a plurality of nucleic acid templates from the dissociated sample, wherein the nucleic acid templates comprise ribonucleic acids; (b) synthesizing complementary deoxyribonucleic acid (cDNA) molecules from the plurality of nucleic acid templates from the dissociated sample, wherein the cDNA molecules are synthesized using a non-naturally occurring R2 enzyme having a processivity of 20 nucleotides or longer, wherein the synthesis is performed in the presence of one or more reagents used in the lysis reaction or in the presence of a plurality of cell fragments from the tissue sample.

49. The method of claim 48, wherein the reaction is performed at a temperature sufficient to drive the dissolution reaction.

50. The method of claim 48, wherein the lysis reaction is substantially enzyme driven.

51. The method of claim 50, wherein the enzyme is selected from collagenase, hyaluronidase, DNase, elastase, papain, protease type XIV, trypsin or lipase.

52. The method of claim 48, wherein the dissolution reaction is performed in the presence of a detergent.

53. The method of claim 48, wherein the non-naturally occurring R2 enzyme synthesizes the cDNA molecule in the presence of the detergent.

54. The method of claim 48, further comprising before step (b) (b) incorporating a first adapter molecule into the 3' end of the plurality of nucleic acid templates from the dissociated sample and incorporating a second adapter molecule into the 5' end of the plurality of nucleic acid templates from the dissociated sample.

55. The method of claim 54, further comprising fragmenting the plurality of nucleic acid templates from the dissociated sample prior to step (b).

56. The method of claim 55, further comprising removing one or more fragmented nucleic acids comprising a 2'-3' cyclic phosphate prior to step (b).

57. The method of claim 56, further comprising adding poly A to the plurality of nucleic acid templates from the dissociated sample prior to step (b).

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